Immunogenic compositions against influenza

ABSTRACT

Disclosed herein are compositions and methods related to mutant viruses, and in particular, mutant influenza viruses. The mutant viruses disclosed herein include mutant M2 sequences, mutant BM2 sequences, and are useful in immunogenic compositions, e.g., as a quadrivalent vaccines. Also disclosed herein are methods, compositions and cells for propagating the viral mutants, and methods, devices and compositions related to vaccination.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a U.S. National Phase Application under 35U.S.C. § 371 of International Application No. PCT/US2018/019653, filedFeb. 26, 2018, which claims the benefit of and priority to U.S.Application No. 62/464,019, filed Feb. 27, 2017, the content of which isincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 27, 2018, isnamed 090248-0163_SL.txt and is 18,149 bytes in size.

BACKGROUND

Influenza is a leading cause of death among American adults. Each year,about 36,000 people die from influenza, and more than 200,000 people arehospitalized. Influenza is a highly contagious disease that is spread bycoughing, sneezing and through direct physical contact with objects thatcarry the virus such as doorknobs and telephones. Symptoms of influenzainclude fever, extreme fatigue, headache, chills and body aches; about50 percent of infected people have no symptoms but are still contagious.Immunization is 50-60 percent effective in preventing influenza inhealthy people under the age of 65, as long as the antigenicities of thecirculating virus strain match those of the vaccine.

Vaccination is the main method for preventing influenza, and both liveattenuated and inactivated (killed) virus vaccines are currentlyavailable. Live virus vaccines, typically administered intranasally,activate all phases of the immune system and can stimulate an immuneresponse to multiple viral antigens. Thus, the use of live virusesovercomes the problem of destruction of viral antigens that may occurduring preparation of inactivated viral vaccines. In addition, theimmunity produced by live virus vaccines is generally more durable, moreeffective, and more cross-reactive than that induced by inactivatedvaccines, and live virus vaccines are less costly to produce thaninactivated virus vaccines. However, the mutations in attenuated virusare often ill-defined, and reversion is a concern.

SUMMARY

In one aspect, the present disclosure provides an immunogeniccomposition, wherein the composition is a multivalent compositioncomprising recombinant viruses from at least two influenza strains.

In some embodiments, the multivalent composition comprises: a) at leastone engineered attenuated influenza A M2-deficient recombinant virus,wherein the engineered influenza A virus comprises a mutant M2 genecomprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and b) at leastone engineered attenuated influenza BM2-deficient recombinant virus,wherein the engineered influenza B virus comprises a mutant BM2 genecomprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, or SEQ ID NO: 11. In some embodiments, the at least oneinfluenza A virus is chosen from the group of H1N1 and H3N2 subtypes,and the at least one influenza B virus is chosen from the group ofB/Yamagata and B/Victoria lineages.

In some embodiments, the multivalent composition comprises recombinantviruses selected from the group consisting of: a) two engineeredattenuated influenza A M2-deficient viruses chosen from the group ofH1N1 and H3N2 subtypes, wherein the A M2-deficient viruses comprise amutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3,and two engineered attenuated influenza BM2-deficient viruses chosenfrom the group of B/Yamagata and B/Victoria lineages, wherein theBM2-deficient viruses comprise a mutant BM2 gene comprising SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ IDNO:11; b) two engineered attenuated influenza A M2-deficient viruseschosen from the group of H1N1 and H3N2 subtypes, wherein the AM2-deficient viruses comprise a mutant M2 gene comprising SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3, and one engineered attenuated influenzaBM2-deficient virus chosen from the group of B/Yamagata and B/Victorialineages, wherein the BM2-deficient virus comprises a mutant BM2 genecomprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, or SEQ ID NO:11; c) one engineered attenuated influenza AM2-deficient virus chosen from the group of H1N1 and H3N2 subtypes,wherein the A M2-deficient virus comprises a mutant M2 gene comprisingSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and two engineeredattenuated influenza BM2-deficient viruses chosen from the group ofB/Yamagata and B/Victoria lineages, wherein the BM2-deficient virusescomprise a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:11; and d) oneengineered attenuated influenza A M2-deficient virus chosen from thegroup of H1N1 and H3N2 subtypes, wherein the A M2-deficient viruscomprises a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3, and one engineered attenuated influenza BM2-deficient viruschosen from the group of B/Yamagata and B/Victoria lineages, wherein theBM2-deficient virus comprises a mutant BM2 gene comprising SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ IDNO:11.

In some embodiments, the multivalent composition is a quadrivalentcomposition comprising: a) two engineered attenuated influenza A virusesconsisting of: i) H1N1 having a mutant M2 gene comprising SEQ ID NO: 1,and ii) H3N2 having a mutant M2 gene comprising SEQ ID NO: 1; and b) twoengineered attenuated influenza B viruses consisting of: i) B/Victoriahaving a mutant BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11, andii) B/Yamagata having a mutant BM2 gene comprising SEQ ID NO: 9 or SEQID NO: 11.

In some embodiments, the multivalent composition is a quadrivalentcomposition comprising: a) two engineered attenuated influenza A virusesconsisting of: i) H1N1 having a mutant M2 gene comprising SEQ ID NO: 1,and ii) H3N2 having a mutant M2 gene comprising SEQ ID NO: 1; and b) oneengineered attenuated influenza B viruses selected from the groupconsisting of: i) B/Victoria having a mutant BM2 gene comprising SEQ IDNO: 9 or SEQ ID NO: 11, and ii) B/Yamagata having a mutant BM2 genecomprising SEQ ID NO: 9 or SEQ ID NO: 11.

In some embodiments, the immunogenic compositions of the presentdisclosure further comprise a pharmaceutically acceptable carrier. Insome embodiments, the immunogenic compositions of the present disclosurefurther comprise a pharmaceutically acceptable adjuvant. In someembodiments, the immunogenic compositions of the present technology areformulated for intranasal or intracutaneous administration.

In one aspect, the present disclosure provides a method of stimulatingan immune response against influenza A and influenza B comprisingadministering to a subject in need thereof a multivalent immunogeniccomposition comprising: a) at least one engineered attenuated influenzaA M2-deficient recombinant virus, wherein the engineered influenza Avirus comprises a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2,or SEQ ID NO: 3; and b) at least one engineered attenuated influenzaBM2-deficient recombinant virus, wherein the engineered influenza Bvirus comprises a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

In some embodiments, the at least one influenza A virus is chosen fromthe group of H1N1 and H3N2 subtypes, and the at least one influenza Bvirus is chosen from the group of B/Yamagata and B/Victoria lineages.

In some embodiments, the multivalent immunogenic composition comprisesrecombinant viruses selected from the group consisting of: a) twoengineered attenuated influenza A M2-deficient viruses chosen from thegroup of H1N1 and H3N2 subtypes, wherein the A M2-deficient virusescomprise a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3, and two engineered attenuated influenza BM2-deficient viruseschosen from the group of B/Yamagata and B/Victoria lineages, wherein theBM2-deficient viruses comprise a mutant BM2 gene comprising SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ IDNO:11; b) two engineered attenuated influenza A M2-deficient viruseschosen from the group of H1N1 and H3N2 subtypes, wherein the AM2-deficient viruses comprise a mutant M2 gene comprising SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3, and one engineered attenuated influenzaBM2-deficient virus chosen from the group of B/Yamagata and B/Victorialineages, wherein the BM2-deficient virus comprises a mutant BM2 genecomprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, or SEQ ID NO:11; c) one engineered attenuated influenza AM2-deficient virus chosen from the group of H1N1 and H3N2 subtypes,wherein the A M2-deficient virus comprises a mutant M2 gene comprisingSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and two engineeredattenuated influenza BM2-deficient viruses chosen from the group ofB/Yamagata and B/Victoria lineages, wherein the BM2-deficient virusescomprise a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:11; and d) oneengineered attenuated influenza A M2-deficient virus chosen from thegroup of H1N1 and H3N2 subtypes, wherein the A M2-deficient viruscomprises a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3, and one engineered attenuated influenza BM2-deficient viruschosen from the group of B/Yamagata and B/Victoria lineages, wherein theBM2-deficient virus comprises a mutant BM2 gene comprising SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ IDNO:11.

In some embodiments, the multivalent immunogenic composition is aquadrivalent composition comprising: a) two engineered attenuatedinfluenza A viruses consisting of: i) H1N1 having a mutant M2 genecomprising SEQ ID NO: 1, and ii) H3N2 having a mutant M2 gene comprisingSEQ ID NO: 1; and b) two engineered attenuated influenza B virusesconsisting of: i) B/Victoria having a mutant BM2 gene comprising SEQ IDNO: 9 or SEQ ID NO: 11, and ii) B/Yamagata having a mutant BM2 genecomprising SEQ ID NO: 9 or SEQ ID NO: 11.

In some embodiments, the multivalent immunogenic composition is aquadrivalent composition comprising: a) two engineered attenuatedinfluenza A viruses consisting of: i) H1N1 having a mutant M2 genecomprising SEQ ID NO: 1, and ii) H3N2 having a mutant M2 gene comprisingSEQ ID NO: 1; and b) one engineered attenuated influenza B virusesselected from the group consisting of: i) B/Victoria having a mutant BM2gene comprising SEQ ID NO: 9 or SEQ ID NO: 11, and ii) B/Yamagata havinga mutant BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11.

In some embodiments, the immunogenic compositions of the presentdisclosure further comprise a pharmaceutically acceptable carrier. Insome embodiments, the immunogenic compositions of the present disclosurefurther comprise a pharmaceutically acceptable adjuvant. In someembodiments, the immunogenic compositions of the present disclosure areformulated for intranasal or intracutaneous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic depicting the role of M2 ion channel in an influenzavirus life cycle, wherein (1) the influenza virus attaches to sialicacid receptors on a cell surface; (2) the virus is internalized into thecell; (3) the M2 ion channel is expressed on the viral surface; (4) theM2 ion channel opens to permit proton entry, leading to a release ofviral RNA that enters the nucleus, is replicated and results in viralprotein synthesis; and (5) the viral components are packaged intovirions and released (6).

FIG. 2 is a chart showing the anti-HA IgG antibody response elicited byBM2SR viruses in a quadrivalent vaccine formulation.

FIGS. 3A-3D are charts showing the anti-HA IgG antibody responseselicited by BM2SR and M2SR viruses in multivalent formulations. Mono/BWI01=monovalent B/WI01 (comprising a BM2SR-0 mutant comprising SEQ IDNO: 11); mono B/Bris60=monovalent B/Bris60 (comprising a BM2SR-0 mutantcomprising SEQ ID NO: 11); mono CA07=monovalent CA07 (comprising anM2SR-1 mutant comprising SEQ ID NO: 1); mono Bris10=monovalent Bris 10(comprising an M2SR-1 mutant comprising SEQ ID NO: 1); BIV H1H3=bivalentH1H3 (comprising M2SR-1 mutants comprising SEQ ID NO: 1); TIVH3VY=trivalent H3VY (comprising M2SR-1 mutant comprising SEQ ID NO: 1and BM2SR-0 mutants comprising SEQ ID NO: 11); TIV H1VY=trivalent H1VY(comprising M2SR-1 mutants comprising SEQ ID NO: 1, and BM2SR-0 mutantcomprising SEQ ID NO: 11); TIV H1H3V=trivalent H1H3V (comprising M2SR-1mutants comprising SEQ ID NO: 1, and BM2SR-0 mutant comprising SEQ IDNO: 11); TIV H1H3Y=trivalent H1H3Y (comprising M2SR-1 mutants comprisingSEQ ID NO: 1, and BM2SR-0 mutant comprising SEQ ID NO: 11); Quadrivalent(comprising M2SR-1 mutants comprising SEQ ID NO: 1, and BM2SR-0 mutantscomprising SEQ ID NO: 11).

FIG. 4A is a chart showing change in mouse body weight after influenza Bchallenge, post-inoculation with monovalent BM2SR, monovalent M2SR, andquadrivalent vaccines.

FIG. 4B is a chart showing mouse survival after influenza B challenge,post-inoculation with monovalent BM2SR, monovalent M2SR, andquadrivalent vaccines.

FIG. 4C is a chart showing the virus titers of mice from differentvaccination groups at Day 4 post influenza B challenge.

FIG. 5A is a chart showing change in mouse body weight after influenza Achallenge, post-inoculation with monovalent BM2SR, monovalent M2SR, andquadrivalent vaccines.

FIG. 5B is a chart showing mouse survival after influenza A challenge,post-inoculation with monovalent BM2SR, monovalent M2SR, andquadrivalent vaccines.

FIG. 5C is a chart showing the virus titers of mice from differentvaccination groups at day 4 post influenza A challenge.

FIGS. 6A-6B are charts showing anti-HA IgG antibody titers elicitedagainst influenza B virus post-inoculation with monovalent BM2SR andquadrivalent formulation. Representative BM2SR constructs: B/CA12 is BYamagata BM2SR; B/Bris46 is B Victoria BM2SR. The quadrivalentformulation is a mix of the two BM2SR and H1N1 and H3N2 M2SR.

FIGS. 7A-7D are charts showing M2SR and BM2SR mutants elicit antibodyresponses against influenza A and influenza B viruses in multivalentformulations. Representative M2SR and BM2SR constructs: A/MA15 is H1N1M2SR; A/HK4801 is H3N2 M2SR; B/CA12 is B Yamagata BM2SR-4; B/Bris46 is BVictoria BM2SR-4. The quadrivalent (“quad”) formulation is a mix of allfour. The trivalent (“tri”) is a formulation of the indicated threeviruses.

FIGS. 8A-8B are charts showing mouse body weight change and survival,respectively, after a lethal dose influenza B challenge,post-inoculation with monovalent BM2SR, trivalent, and quadrivalentformulations.

FIGS. 9A-9B are charts showing mouse body weight change and survival,respectively, after a lethal dose influenza A challenge,post-inoculation with trivalent M2SR formulation (comprising H1N1comprising an M2SR-1 mutant comprising SEQ ID NO: 1), H3N2 (comprisingan M2SR-1 mutant comprising SEQ ID NO: 1), and B/Yamagata (comprising aBM2SR-4 mutant comprising SEQ ID NO: 9)) or a quadrivalent M2SRformulation (comprising H1N1 (comprising H1N1 (comprising an M2SR-1mutant comprising SEQ ID NO: 1), H3N2 (comprising an M2SR-1 mutantcomprising SEQ ID NO: 1), B/Victoria lineage (comprising a BM2SR-4mutant comprising SEQ ID NO: 9, and B/Yamagata (comprising a BM2SR-4mutant comprising SEQ ID NO: 9)).

FIG. 10 is a chart showing enzyme-linked immunosorbent assay (ELISA)titers elicited against each component of a quadrivalent vaccine.

FIG. 11A is a chart showing hemagglutination inhibition (HAI) titerselicited against each component of a quadrivalent vaccine.

FIG. 11B is a table showing HAI titers elicited against each componentof a quadrivalent vaccine in pooled sera at day 35 post inoculation.

FIG. 12 is a chart showing nasal wash virus titers on days 1, 3, 5, and7 post influenza A challenge.

FIG. 13A is a chart showing virus titer in nasal turbinate tissue on day3 post influenza A challenge.

FIG. 13B is a chart showing virus titer in trachea tissue on day 3 postinfluenza A challenge.

FIG. 13C is a chart showing virus titer in lung tissue on day 3 postinfluenza A challenge.

DETAILED DESCRIPTION I. Definitions

The following terms are used herein, the definitions of which areprovided for guidance.

As used herein, the singular forms “a,” “an,” and “the” designate boththe singular and the plural, unless expressly stated to designate thesingular only.

The term “about” and the use of ranges in general, whether or notqualified by the term about, means that the number comprehended is notlimited to the exact number set forth herein, and is intended to referto ranges substantially within the quoted range while not departing fromthe scope of the invention. As used herein, “about” will be understoodby persons of ordinary skill in the art and will vary to some extent onthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art given the context inwhich it is used, “about” will mean up to plus or minus 10% of theparticular term.

As used herein, the term “attenuated,” as used in conjunction with avirus, refers to a virus having reduced virulence or pathogenicity ascompared to a non-attenuated counterpart, yet is still viable or live.Typically, attenuation renders an infectious agent, such as a virus,less harmful or virulent to an infected subject compared to anon-attenuated virus. This is in contrast to killed or completelyinactivated virus.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” or “pharmaceutically effective amount” refer to aquantity sufficient to achieve a desired therapeutic and/or prophylacticeffect, e.g., an amount which results in the prevention of, disease,condition and/or symptom(s) thereof. In the context of therapeutic orprophylactic applications, the amount of a composition administered tothe subject will depend on the type and severity of the disease and onthe characteristics of the individual, such as general health, age, sex,body weight and tolerance to the composition drugs. It will also dependon the degree, severity and type of disease or condition. The skilledartisan will be able to determine appropriate dosages depending on theseand other factors. In some embodiments, multiple doses are administered.Additionally or alternatively, in some embodiments, multiple therapeuticcompositions or compounds (e.g., immunogenic compositions, such asvaccines) are administered.

As used herein, the term “host cell” refers to a cell in which apathogen, such as a virus, can replicate. In some embodiments, hostcells are in vitro, cultured cells. Non-limiting examples of such hostcells include, but are not limited to, CHO cells, Vero cells, and MDCKcells. Additionally or alternatively, in some embodiments, host cellsare in vivo (e.g., cells of an infected vertebrate, such as an avian ormammal). In some embodiments, the host cells may be modified, e.g., toenhance viral production such as by enhancing viral infection of thehost cell and/or by enhancing viral growth rate. By way of example, butnot by way of limitation, exemplary host cell modifications includerecombinant expression of 2-6-linked sialic acid receptors on the cellsurface of the host cell, and/or recombinant expression of a protein inthe host cells that has been rendered absent or ineffective in thepathogen or virus.

The term “immunogenic composition” is used herein to refer to acomposition that will elicit an immune response in a mammal that hasbeen exposed to the composition. In some embodiments, an immunogeniccomposition comprises at least one M2-deficient mutant influenza A M2SRstrain (e.g., A/California/07/2009 (H1N1) (comprising an M2SR mutantcomprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3),A/Brisbane/10/2007 (H3N2) (comprising an M2SR mutant comprising SEQ IDNO: 1, SEQ ID NO: 2, or SEQ ID NO: 3). In some embodiments, animmunogenic composition comprises at least one BM2-deficient mutantinfluenza B BM2SR strain (e.g., B/Brisbane/60/2008 (Victoria)(comprising a BM2SR mutant comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11),B/Wisconsin/01/2010 (Yamagata) (comprising a BM2SR mutant comprising SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, orSEQ ID NO: 11). In some embodiments, an immunogenic compositioncomprises A/California/07/2009 (H1N1) (comprising an M2SR mutantcomprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3),A/Brisbane/10/2007 (H3N2) (comprising an M2SR mutant comprising SEQ IDNO: 1, SEQ ID NO: 2, or SEQ ID NO: 3), B/Brisbane/60/2008 (Victoria)(comprising a BM2SR mutant comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11), andB/Wisconsin/01/2010 (Yamagata) (comprising a BM2SR mutant comprising SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, orSEQ ID NO: 11) formulated as a quadrivalent vaccine.

In some embodiments, the immunogenic compositions described herein maybe formulated for administration (i.e., formulated for “exposure” to themammal) in a number of forms. For example, in some embodiments, theimmunogenic compositions are prepared for oral, pulmonary, intravenous,intramuscular, subcutaneous, parenteral, nasal, or topicaladministration. Compositions may also be formulated for specific dosageforms. For example, in some embodiments, the immunogenic composition maybe formulated as a liquid, gel, aerosol, ointment, cream, lyophilizedformulation, powder, cake, tablet, or capsule. In other embodiments, theimmunogenic composition is formulated as a controlled releaseformulation, delayed release formulation, extended release formulation,pulsatile release formulation, and mixed immediate release formulation.In some embodiments, the immunogenic composition is provided as aliquid. In other embodiments, the immunogenic composition is provided inlyophilized form.

As used herein, the term “infected” refers to harboring a disease orpathogen, such as a virus. An infection can be intentional, such as byadministration of a virus or pathogen (e.g., by vaccination), orunintentional, such as by natural transfer of the pathogen from oneorganism to another, or from a contaminated surface to the organism.

As used herein, the terms “isolated” and/or “purified” refer to in vitropreparation, isolation and/or purification of a nucleic acid (e.g., avector or plasmid), polypeptide, virus or cell such that it is notassociated with unwanted in vivo substances, or is substantiallypurified from unwanted in vivo substances with which it normally occurs.For example, in some embodiments, an isolated virus preparation isobtained by in vitro culture and propagation, and is substantially freefrom other infectious agents. As used herein, “substantially free” meansbelow the level of detection for a particular compound, such as unwantednucleic acids, proteins, cells, viruses, infectious agents, etc. usingstandard detection methods for that compound or agent.

As used herein the terms “mutant,” “mutation,” and “variant” are usedinterchangeably and refer to a nucleic acid or polypeptide sequencewhich differs from a wild-type sequences. In some embodiments, mutant orvariant sequences are naturally occurring. In other embodiments, mutantor variant sequences are recombinantly and/or chemically introduced. Insome embodiments, nucleic acid mutations include modifications (e.g.,additions, deletions, substitutions) to RNA and/or DNA sequences. Insome embodiments, modifications include chemical modification (e.g.,methylation) and may also include the substitution or addition ofnatural and/or non-natural nucleotides. Nucleic acid mutations may besilent mutations (e.g., one or more nucleic acid changes which code forthe same amino acid as the wild-type sequence) or may result in a changein the encoded amino acid, result in a stop codon, or may introducesplicing defects or splicing alterations. Nucleic acid mutations tocoding sequences may also result in conservative or non-conservativeamino acid changes.

As used herein the term “recombinant virus” refers to a virus that hasbeen manipulated in vitro, e.g., using recombinant nucleic acidtechniques, to introduce changes to the viral genome and/or to introducechanges to the viral proteins. For example, in some embodiments,recombinant viruses may include both wild-type, endogenous, nucleic acidsequences and mutant and/or exogenous nucleic acid sequences.Additionally or alternatively, in some embodiments, recombinant virusesmay include modified protein components, such as mutant or variantmatrix, hemagglutinin, neuraminidase, nucleoprotein, non-structuraland/or polymerase proteins.

As used herein the term “recombinant cell” or “modified cell” refer to acell that has been manipulated in vitro, e.g., using recombinant nucleicacid techniques, to introduce nucleic acid into the cell and/or tomodify cellular nucleic acids. Examples of recombinant cells includesprokaryotic or eukaryotic cells carrying exogenous plasmids, expressionvectors and the like, and/or cells which include modifications to theircellular nucleic acid (e.g., substitutions, mutations, insertions,deletions, etc., into the cellular genome). An exemplary recombinantcell is one which has been manipulated in vitro to stably express anexogenous protein, such as a viral M2 protein.

As used herein the term “single replication (SR) virus” refers to avirus that is defective in a virion protein that functions in viralentry of a host cell or release from a host cell. For example, M2SR, asdescribed herein, belongs to the novel class of single-replication (SR)virus vaccines in contrast to classical live attenuated influenzavaccines. SR viruses are defective in a virion protein that functions inviral entry or release, such as the flu M2 ion channel protein that doesnot affect viral genome replication but is indispensable for virusgrowth. In contrast, traditional attenuated live virus vaccines containmultiple mutations in the viral replication machinery resulting in ahighly attenuated phenotype. The SR vaccine virus mechanisms thereforedo not affect the virus infection kinetics and antigen production incontrast to attenuated live vaccines.

As used herein “subject” and “patient” are used interchangeably andrefer to an animal, for example, a member of any vertebrate species. Themethods and compositions of the presently disclosed subject matter areparticularly useful for warm-blooded vertebrates including mammals andbirds. Exemplary subjects may include mammals such as humans, as well asmammals and birds of importance due to being endangered, of economicimportance (animals raised on farms for consumption by humans) and/or ofsocial importance (animals kept as pets or in zoos) to humans. In someembodiments, the subject is a human. In some embodiments, the subject isnot human.

As used herein, the term “type” and “strain” as used in conjunction witha virus are used interchangeably, and are used to generally refer toviruses having different characteristics. For example, influenza A virusis a different type of virus than influenza B virus. Likewise, influenzaA H1N1 is a different type of virus than influenza A H2N1, H2N2 andH3N2. Additionally or alternatively, in some embodiments, differenttypes of virus such as influenza A H2N1, H2N2 and H3N2 may be termed“subtypes.”

The term “vaccine” is used herein to refer to a composition that isadministered to a subject to produce or increase immunity to aparticular disease. In some embodiments, vaccines include apharmaceutically acceptable adjuvant and/or a pharmaceuticallyacceptable carrier.

As used herein, the term “vRNA” refers to the RNA comprising a viralgenome, including segmented or non-segmented viral genomes, as well aspositive and negative strand viral genomes. vRNA may be whollyendogenous and “wild-type” and/or may include recombinant and/or mutantsequences.

The term “virulence” is used herein to refer to the relative ability ofa pathogen to cause disease.

The term “attenuated virulence” or “reduced virulence” is used herein torefer to a reduced relative ability of a pathogen to cause disease. Forexample, attenuated virulence or reduced virulence can describe virusesthat have been weakened so they produce immunity when exposed to asubject, but do not cause disease, or cause a less severe form,duration, onset or later onset of the disease.

As used herein, “M2SR” refers to a single-replication (SR) M2-deficientrecombinant influenza virus. Exemplary M2SR influenza viruses describedherein comprise SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3, a viruscomprising SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3, or a vaccinecomprising a virus comprising SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ IDNO: 3, depending on the context in which it is used. For example, indescribing mutations of the M2 gene demonstrated herein, “M2SR” refersto SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3. In particular,“M2SR-1” refers to SEQ ID NO: 1; “M2SR-2” refers to SEQ ID NO: 2; and“M2SR-3” refers to SEQ ID NO: 3. When describing the viral component ofa vaccine, “M2SR” refers to a recombinant influenza virus that does notexpress functional M2 protein. When describing a vaccine, “M2SR” refersto a vaccine comprising the M2SR recombinant virus.

As used herein, “M2SR virus” encompasses a recombinant influenza virusthat does not express functional M2 protein. In some embodiments, theM2SR virus comprises genes of other influenza viruses. In someembodiments, the virus comprises the HA and NA genes of InfluenzaA/Brisbane/10/2007-like A/Uruguay/716/2007(H3N2). In some embodiments,the M2SR virus comprises the HA and NA genes of the A/California/07/2009(CA07) (H1N1pdm) virus.

As used herein, “BM2SR” refers to a single-replication (SR)BM2-deficient recombinant influenza virus. Exemplary BM2SR influenzaviruses described herein comprise SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, and/or SEQ ID NO: 11, a virus comprisingSEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,and/or SEQ ID NO: 11, or a vaccine comprising a virus comprising SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and/orSEQ ID NO: 11, depending on the context in which it is used. Forexample, in describing mutations of the BM2 gene demonstrated herein,“BM2SR” refers to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, and/or SEQ ID NO: 11. In particular, “BM2SR-1” refersto SEQ ID NO: 6; “BM2SR-2” refers to SEQ ID NO: 7; “BM2SR-3” refers toSEQ ID NO: 8; “BM2SR-4” refers to SEQ ID NO: 9; “BM2SR-5” refers to SEQID NO: 10; and “BM2SR-0” refers to SEQ ID NO: 11. When describing theviral component of a vaccine, “BM2SR” refers to a recombinant influenzavirus which by way of example, but not by way of limitation, possessesinternal genes of B/Lee/40 (nucleoprotein (NP), polymerase genes (PA,PB1, PB2), non-structural (NS1 and NS2), NB, matrix (BM1)), but whichdoes not express functional BM2 protein. When describing a vaccine,“BM2SR” refers to a vaccine comprising the BM2SR recombinant virus.

As used herein, “BM2SR virus” encompasses a recombinant influenza viruswhich possesses internal genes of B/Lee/40 (nucleoprotein (NP),polymerase genes (PA, PB1, PB2), non-structural (NS1 and NS2), matrix(BM1)), but which does not express functional BM2 protein, alone or incombination with other viral components and/or genes encoding otherviral components. In some embodiments, the BM2SR virus comprises genesof other influenza viruses. In some embodiments, the virus comprises theHA and NA genes of Influenza B/Brisbane/60/2008-like B/Brisbane/60/2008(B Victoria lineage). In some embodiments, the M2SR virus comprises theHA and NA genes of the B/Wisconsin/1/2010-like (B Yamagata lineage)virus. In some embodiments, the BM2SR virus possesses internal genes(NP, PA, PB1, PB2, NS1 and NS2, BM1) of recent influenza B viruses.

II. Influenza A Virus and Influenza B Virus

A. General

Influenza is a leading cause of death among American adults. The causalagent of influenza are viruses of the family Orthomyxoviridae includinginfluenza A virus, influenza B virus, and influenza C virus.

The influenza A virus is an enveloped, negative-strand RNA virus. Thegenome of influenza A virus is contained on eight single (non-paired)RNA strands the complements of which code for eleven proteins (HA, NA,NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The total genome size isabout 14,000 bases. The segmented nature of the genome allows for theexchange of entire genes between different viral strains during cellularcohabitation. The eight RNA segments are as follows. 1) HA encodeshemagglutinin (about 500 molecules of hemagglutinin are needed to makeone virion); 2) NA encodes neuraminidase (about 100 molecules ofneuraminidase are needed to make one virion); 3) NP encodesnucleoprotein; 4) M encodes two proteins (the M1 and the M2) by usingdifferent reading frames from the same RNA segment (about 3000 M1molecules are needed to make one virion); 5) NS encodes two proteins(NS1 and NEP) by using different reading frames from the same RNAsegment; 6) PA encodes an RNA polymerase; 7) PB1 encodes an RNApolymerase and PB1-F2 protein (induces apoptosis) by using differentreading frames from the same RNA segment; 8) PB2 encodes an RNApolymerase.

The influenza B virus is also an enveloped, negative-strand RNA virus.The genome of influenza B virus is contained on eight single(non-paired) RNA strands the complements of which code for elevenproteins. Of these proteins, nine are also found in influenza A virus:three RNA-dependent RNA polymerase subunits (PB1, PB2, and PA),hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrixprotein (M1 or BM1), and two non-structural proteins (NS1 and NS2, alsoknown as NEP). Two proteins, NB and BM2, are unique to influenza Bvirus. The total genome size is about 14,500 bases. The segmented natureof the genome allows for the exchange of entire genes between differentviral strains during cellular cohabitation, a process known asreassortment. The eight RNA segments, numbered in order of decreasinglength, are as follows. Segments 1, 2, and 3 encode PB1, PB2, and PA,respectively, which are RNA polymerase subunits. Segment 4 encodes HA(hemagglutinin). Segment 5 encodes NP (nucleoprotein). Segment 6 encodesboth NB (NB protein, the function of which is unknown) and NA(neuraminidase). Segment 7 encodes both BM1 (matrix protein) and BM2(ion channel) by a bicistronic mRNA, the translational strategy of whichis unique. The BM2 initiation codon overlaps with the BM1 terminationcodon (TAATG, a stop-start pentanucleotide motif). The BM2 protein istranslated by this stop-start translational mechanism unlike the M2protein of influenza A virus, which is translated from a spliced mRNA.Segment 8 encodes both NS1 and NEP by using different reading framesfrom the same RNA segment.

Both influenza A and B evolve antigenically over time by the process ofantigenic drift, in which mutations to hemagglutinin (HA) protein allowviruses to escape existing human immunity and persist in the humanpopulation. There are several subtypes of influenza A, named accordingto an H number (for the type of hemagglutinin) and an N number (for thetype of neuraminidase). Currently, there are 16 different H antigensknown (H1 to H16) and nine different N antigens known (N1 to N9). Eachvirus subtype has mutated into a variety of strains with differingpathogenic profiles; some pathogenic to one species but not others, somepathogenic to multiple species. Exemplary Influenza A virus subtypesthat have been confirmed in humans, include, but are not limited to H1N1which caused the “Spanish Flu” and the 2009 swine flu outbreak; H2N2which caused the “Asian Flu” in the late 1950s; H3N2 which caused theHong Kong Flu in the late 1960s; H5N1, considered a global influenzapandemic threat through its spread in the mid-2000s; H7N7; H1N2 which iscurrently endemic in humans and pigs; and H9N2, H7N2, H7N3, H5N2, H10N7.

Two antigenically and genetically distinct lineages of influenza Bviruses have co-circulated and caused disease in humans since at least1988. Influenza viruses of the Victoria lineage were the predominanttype B strains circulating worldwide in the 1980s with the Yamagatalineage becoming the dominant type B virus in the early 1990s. Since1991, Victoria lineage viruses have been isolated infrequently and beenlimited almost entirely to eastern Asia. Victoria viruses reemerged in2002 and both Yamagata and Victoria lineages have coexisted since.

Evolutionary relationships of influenza B viruses isolated from 1940 to2016 indicate that the BM1 and BM2 proteins of modern isolates are moreclosely related to each other than to B/Lee/40.

Influenza viruses have a standard nomenclature that includes virus type;species from which it was isolated (if non-human); location at which itwas isolated; isolate number; isolate year; and, for influenza A virusesonly, HA and NA subtype. Thus, B/Yamagata/16/88 was isolate number 16 ofa human influenza B virus taken in Yamagata (Japan) in 1988.

Some influenza A variants are identified and named according to theknown isolate to which they are most similar, and thus are presumed toshare lineage (e.g., Fujian flu virus-like); according to their typicalhost (example Human flu virus); according to their subtype (exampleH3N2); and according to their pathogenicity (example LP, LowPathogenic). Thus, a flu from a virus similar to the isolateA/Fujian/411/2002(H3N2) can be called Fujian flu, human flu, and H3N2flu.

In addition, influenza variants are sometimes named according to thespecies (host) the strain is endemic in or adapted to. The main variantsnamed using this convention are: bird flu, human flu, swine influenza,equine influenza and canine influenza. Variants have also been namedaccording to their pathogenicity in poultry, especially chickens, e.g.,Low Pathogenic Avian Influenza (LPAI) and Highly Pathogenic AvianInfluenza (HPAI).

B. Life Cycle and Structure

The life cycle of influenza viruses generally involves attachment tocell surface receptors, entry into the cell and uncoating of the viralnucleic acid, followed by replication of the viral genes inside thecell. After the synthesis of new copies of viral proteins and genes,these components assemble into progeny virus particles, which then exitthe cell. Different viral proteins play a role in each of these steps.

The influenza A particle is made up of a lipid envelope whichencapsulates the viral core. The inner side of the envelope is lined bythe matrix protein (M1), while the outer surface is characterized by twotypes of glycoprotein spikes: hemagglutinin (HA) and neuraminidase (NA).M2, a transmembrane ion channel protein, is also part of the lipidenvelope. See e.g., FIG. 1. The influenza B particle comprises a similarstructure.

The HA protein, a trimeric type I membrane protein, is responsible forbinding to sialyloligosaccharides (oligosaccharides containing terminalsialic acid linked to galactose) on host cell surface glycoproteins orglycolipids. This protein is also responsible for fusion between viraland host cell membranes, following virion internalization byendocytosis.

Neuraminidase (NA), a tetrameric type II membrane protein, is asialidase that cleaves terminal sialic acid residues from theglycoconjugates of host cells and the HA and NA, and thus is recognizedas receptor-destroying enzyme. This sialidase activity is necessary forefficient release of progeny virions from the host cell surface, as wellas prevention of progeny aggregation due to the binding activity ofviral HAs with other glycoproteins. Thus, the receptor-binding activityof the HA and the receptor-destroying activity of the NA likely act ascounterbalances, allowing efficient replication of influenza.

The genome segments are packaged into the core of the viral particle.The RNP (RNA plus nucleoprotein, NP) is in helical form with three viralpolymerase polypeptides associated with each segment.

The influenza virus life cycle begins with binding of the HA to sialicacid-containing receptors on the surface of the host cell, followed byreceptor-mediated endocytosis (FIG. 1). The low pH in late endosomestriggers a conformational shift in the HA, thereby exposing theN-terminus of the HA2 subunit (the so-called fusion peptide). The fusionpeptide initiates the fusion of the viral and endosomal membrane, andthe matrix protein (M1) and RNP complexes are released into thecytoplasm. RNPs consist of the nucleoprotein (NP), which encapsidatesvRNA, and the viral polymerase complex, which is formed by the PA, PB1,and PB2 proteins. RNPs are transported into the nucleus, wheretranscription and replication take place. The RNA polymerase complexcatalyzes three different reactions: (1) synthesis of an mRNA with a 5′cap and 3′ polyA structure, (2) a full-length complementary RNA (cRNA),and (3) genomic vRNA using the cDNA as a template. Newly synthesizedvRNAs, NP, and polymerase proteins are then assembled into RNPs,exported from the nucleus, and transported to the plasma membrane, wherebudding of progeny virus particles occurs. The neuraminidase (NA)protein plays a role late in infection by removing sialic acid fromsialyloligosaccharides, thus releasing newly assembled virions from thecell surface and preventing the self-aggregation of virus particles.Although virus assembly involves protein-protein and protein-vRNAinteractions, the nature of these interactions remains largely unknown.

C. Role of the M2 and BM2 Protein

As described above, spanning the influenza A viral membrane are threeproteins: hemagglutinin (HA), neuramimidase (NA), and M2. Theextracellular domains (ectodomains) of HA and NA are quite variable,while the ectodomain domain of M2 is essentially invariant amonginfluenza A viruses. The M2 ion channel protein does not affect viralgenome replication but is indispensable for virus growth. Singlereplication (SR) viruses are defective in a virion protein thatfunctions in viral entry or release, such as the influenza A M2 orinfluenza BM2 ion channel protein. In contrast, traditional attenuatedlive virus vaccines contain multiple mutations in the viral replicationmachinery resulting in a highly attenuated phenotype. Without wishing tobe bound by theory, in influenza A viruses, the M2 protein, whichpossesses ion channel activity, is thought to function at an early statein the viral life cycle between host cell penetration and un-coating ofviral RNA. Once virions have undergone endocytosis, thevirion-associated M2 ion channel, a homotetrameric helix bundle, isbelieved to permit protons to flow from the endosome into the virioninterior to disrupt acid-labile M1 protein-ribonucleoprotein complex(RNP) interactions, thereby promoting RNP release into the cytoplasm. Inaddition, among some influenza strains whose HAs are cleavedintracellularly (e.g., A/fowl plagues/Rostock/34), the M2 ion channel isthought to raise the pH of the trans-Golgi network, preventingconformational changes in the HA due to conditions of low pH in thiscompartment. It was also shown that the M2 transmembrane domain itselfcan function as an ion channel. M2 protein ion channel activity isthought to be essential in the life cycle of influenza viruses, becauseamantadine hydrochloride, which blocks M2 ion channel activity, has beenshown to inhibit viral replication. However, a requirement for thisactivity in the replication of influenza A viruses has not been directlydemonstrated. The functional counterpart to the influenza A virus M2protein in influenza B viruses is the type III transmembrane proteinknown as BM2.

D. M2 and BM2 Viral Mutants as Vaccines

M2SR belongs to the novel class of single-replication (SR) virusvaccines. SR viruses are defective in a virion protein that functions inviral entry or release, such as the flu M2 ion channel protein, that donot affect viral genome replication but are indispensable for virusgrowth. In contrast, traditional live attenuated vaccines containmultiple mutations in the viral replication machinery resulting in ahighly attenuated phenotype. The two different vaccine virus mechanismstherefore affect the virus infection kinetics and antigen production,which affect protection and induction of immune responses.Replication-defective viruses provide unique forms of viral vaccinesthat combine the safety of an inactivated virus vaccine and theimmunogenicity of a live virus vaccine by expressing viral gene productswithin cells so the antigens can be presented efficiently by both MHCclass I and class II pathways. Single replication viruses can alsoactivate Toll-like receptors and other innate immune response pathways,thereby serving as their own adjuvants. In addition, these viruses canbe used as tools to probe the function of the immune system. Thesemutant viruses are defective in a virion protein that functions afterviral assembly. The viruses are propagated in complementing cells thatexpress the missing gene product. In normal cells, the replication cycleoccurs normally and progeny virions are produced. However, these virionsare noninfectious so the infection does not spread to a second round ofcells.

III. M2 and BM2 Viral Mutants

In one aspect, influenza A viruses harboring a mutant M2 vRNA sequenceare disclosed. Typically, such mutants do not have M2 ion channelactivity, exhibit attenuated growth properties in vivo, cannot produceinfectious progeny and are non-pathogenic or show reduced pathogenesisin infected subjects. In another aspect, influenza B viruses harboring amutant BM2 vRNA sequence are disclosed. Typically, such mutants do nothave BM2 ion channel activity, exhibit attenuated growth properties invivo, cannot produce infectious progeny and are non-pathogenic or showreduced pathogenesis in infected subjects. The mutant viruses areimmunogenic, and when used as a vaccine, provide protection againstinfection with a counterpart wild-type and/or other pathogenic virus.Additionally, the M2 and BM2 mutants disclosed herein are stable, and donot mutate to express a functional M2 or BM2 polypeptide, regardless ofthe host cell used. Additionally or alternatively, in some embodiments,the M1 protein of these mutants is produced without detectablealteration to its function. In some embodiments, viruses harboring themutant M2 or BM2 nucleic acid sequences cannot replicate in a host cellin which a corresponding wild-type virus could be propagated. By way ofexample, but not by way of limitation, in some embodiments, thewild-type virus can be grown, propagated and replicate in culturing MDCKcells, CHO cells and/or Vero cells, while the corresponding virusharboring a mutant M2 or BM2 sequence cannot grow, replicate or bepropagated in the same type of cells.

As noted above, in some embodiments, the M2 or BM2 mutant virus isstable, and does not mutate or revert to wild-type or to a non-wild-typesequence encoding a functional M2 or BM2 protein in a host cell. Forexample, in some embodiments, the M2 or BM2 mutant virus is stable for 2passages, 3 passages, 5 passages, 10 passages, 12 passages, 15 passages,20 passages, 25 passages or more than 25 passages in a host cell. Insome embodiments, the host cell is an unmodified host cell. In someembodiments, the host cell is any mammalian cell stably providing M2 intrans. In other embodiments, the host cell is a modified host cell, suchas a MDCK or Vero cell which expresses the M2 or BM2 protein.

In some embodiments, the M2 or BM2 mutants include one or more nucleicacid substitutions and/or deletions. In some embodiments, the mutationsare localized in nucleic acids which code for one or more of theextracellular domain of the M2 or BM2 protein, the transmembrane domainof the M2 or BM2 proteins, and/or the cytoplasmic tail of the M2 or BM2protein. Additionally or alternatively, in some embodiments, one or morenucleic acid mutations results in a splice variant, one or more stopcodons and/or one or more amino acid deletions of the M2 or BM2 peptide.In some embodiments, viruses carrying the mutant M2 or BM2 nucleic acidproduce a non-functional M2 or BM2 polypeptide. In some embodiments,viruses carrying the mutant M2 or BM2 nucleic acid do not produce an M2or BM2 polypeptide. In some embodiments, viruses carrying the mutant M2or BM2 nucleic acid produce a truncated M2 or BM2 polypeptide.

Three exemplary, non-limiting M2 viral mutants (M2SR-1, M2SR-2 andM2SR-3) are provided below in Tables 1-3. In the tables, lower caseletters correspond to the M2 sequence; upper case letters correspond tothe M1 sequence and non-coding regions; mutant sequence (e.g., stopcodons, splice defect) are in bold, underlined. Underlined (lower case)bases in the M2SR-2 mutant indicate the region deleted in the M2SR-1 andM2SR-3 mutants. Lower case italicized bases include M and M2 overlapregions.

TABLE 1 M2SR-1 (SEQ ID NO: 1) M2 ectodomain +2 stop codons + TM deletion (PR8 M segment +2 stops (786-791) without 792-842 (TM)).3′ AGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgaggtcgaaacGTACGTACTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGATCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTCAAGAGGGAGATAACATTCCATGGGGCCAAAGAAATCTCACTCAGTTATTCTGCTGGTGCACTTGCCAGTTGTATGGGCCTCATATACAACAGGATGGGGGCTGTGACCACTGAAGTGGCATTTGGCCTGGTATGTGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAAATGGTGACAACAACCAATCCACTAATCAGACATGAGAACAGAATGGTTTTAGCCAGCACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAAGCAGCAGAGGCCATGGAGGTTGCTAGTCAGGCTAGACAAATGGTGCAAGCGATGAGAACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCTGAAAAATGATCTTCTTGAAAATTTGCAGgcctatcagaaacgaatgggggtgcagatgcaacggttca agtgat TAATAGgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggccttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatggtcattttgtcagcatagagctggagtaaAAAACTA CCTTGTTTCTACT

The M2 polypeptide sequence produced from this mutant is as follows:

(SEQ ID NO: 4) MSLLTEVETPIRNEWGCRCNGSSD..

TABLE 2 M2SR-2 (SEQ ID NO: 2) M2 ectodomain + 2 stops + splice defect (PR8 M segment + 2 stops (786-791) + splice defect nt 52).3′ AGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgag gtcgaaac CTACGTACTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGATCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTCAAGAGGGAGATAACATTCCATGGGGCCAAAGAAATCTCACTCAGTTATTCTGCTGGTGCACTTGCCAGTTGTATGGGCCTCATATACAACAGGATGGGGGCTGTGACCACTGAAGTGGCATTTGGCCTGGTATGTGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAAATGGTGACAACAACCAATCCACTAATCAGACATGAGAACAGAATGGTTTTAGCCAGCACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAAGCAGCAGAGGCCATGGAGGTTGCTAGTCAGGCTAGACAAATGGTGCAAGCGATGAGAACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCTGAAAAATGATCTTCTTGAAAATTTGCAGgcctatcagaaacgaatgggggtgcagatgcaacggttca agtgat TAATAGactattgccgcaaatatcattgggatcttgcacttgacattgtggattcttgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggccttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatggtcattttgtcagcatagagctggagtaaAA AACTACCTTGTTTCTACT

No M2 polypeptide sequence is produced from this mutant.

TABLE 3 M2SR-3 (SEQ ID NO: 3) M2 ectodomain +2 stops + splice defect + TM deletion (PR8 M segment + 2 stops(786-791) without 792-842 (TM) + splice defect nt 52).3′ AGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgag gtcgaaac CTACGTACTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGATCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTCAAGAGGGAGATAACATTCCATGGGGCCAAAGAAATCTCACTCAGTTATTCTGCTGGTGCACTTGCCAGTTGTATGGGCCTCATATACAACAGGATGGGGGCTGTGACCACTGAAGTGGCATTTGGCCTGGTATGTGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAAATGGTGACAACAACCAATCCACTAATCAGACATGAGAACAGAATGGTTTTAGCCAGCACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAAGCAGCAGAGGCCATGGAGGTTGCTAGTCAGGCTAGACAAATGGTGCAAGCGATGAGAACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCTGAAAAATGATCTTCTTGAAAATTTGCAGgcctatcagaaacgaatgggggtgcagatgcaacggttca agtgat TAATAGgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggccttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatggtcattttgtcagcatagagctggagtaaAAAACTA CCTTGTTTCTACT

No M2 polypeptide sequence is produced from this mutant.

The wild-type M1 and M2 coding sequence is provided below in Table 4.

TABLE 4 M1/M2 wild-type nucleic acid sequence (SEQ ID NO: 5)3′ AGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgaggtcgaaacGTACGTACTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGATCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTCAAGAGGGAGATAACATTCCATGGGGCCAAAGAAATCTCACTCAGTTATTCTGCTGGTGCACTTGCCAGTTGTATGGGCCTCATATACAACAGGATGGGGGCTGTGACCACTGAAGTGGCATTTGGCCTGGTATGTGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAAATGGTGACAACAACCAATCCACTAATCAGACATGAGAACAGAATGGTTTTAGCCAGCACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAAGCAGCAGAGGCCATGGAGGTTGCTAGTCAGGCTAGACAAATGGTGCAAGCGATGAGAACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCTGAAAAATGATCTTCTTGAAAATTTGCAGgcctatcagaaacgaatgggggtgcagatgcaacggttcaagtgatcctctcactattgccgcaaatatcattgggatcttgcacttgacattgtggattcttgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggccttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatggtcattttgtcagcatagagctggagtaaAA AACTACCTTGTTTCTACT

Exemplary, non-limiting BM2SR viral mutants (BM2SR-1, BM2SR-2, BM2SR-3,BM2SR-4, BM2SR-5, and BM2SR-0) are provided below in Table 5.

TABLE 5 BM2SR Sequences M segment 7 sequences of BM2SR influenzaviruses containing null mutations in BM2 genes.BM2SR-1 (SEQ ID NO: 6) influenza B/FL/4/2006Segment 7 with intact BM1 + total BM2 deletionof 329 bp (indicated by -) (mRNA sense).5′ AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCATTGACAGAAGATGGAGAAGGCAAAGCAGAACTAGCAGAAAAATTACACTGTTGGTTCGGTGGGAAAGAATTTGACCTAGACTCTGCCTTGGAATGGATAAAAAACAAAAGATGCTTAACTGACATACAGAAAGCACTAATTGGCGCCTCTATCTGCTTTTTAAAACCCAAAGACCAGGAAAGAAAAAGAAGATTCATCACAGAGCCCCTATCAGGAATGGGGACAACAGCAACAAAAAAGAAGGGCCTGATTCTAGCTGAGAGAAAAATGAGAAGATGTGTGAGCTTCCATGAAGCATTTGAAATAGCAGAAGGCCATGAAAGCTCAGCGTTACTATATTGTCTCATGGTCATGTACCTGAATCCTGGAAATTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTGTGCGAAAAACAAGCATCACATTCACACAGGGCTCATAGCAGAGCAGCGAGATCTTCAGTGCCTGGAGTGAGACGGGAAATGCAGATGGTCTCAGCTATGAACACAGCAAAAACAATGAATGGAATGGGAAAAGGAGAAGACGTTCAAAAACTGGCAGAAGAACTGCAAAGCAACATTGGAGTATTGAGATCTCTTGGGGCAAGTCAAAAGAATGGGGAAGGAATTGCAAAGGATGTAATGGAAGTGCTAAAGCAGAGCTCTATGGGAAATTCAGCTCTTGTGAAGAAATACCTATAA-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ATTCAATTTTTACTGTACTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGCAAATAAACTGGAAAAAGTGCGTTGTTTCTACTBOLD UPPER CASE = BM1 ORF Stop Codon - = designates deleted nucleotidesBM2SR-2 (SEQ ID NO: 7) influenza B/FL/4/2006 Segment 7 with intact BM1 + partial BM2 deletion of 296 bp (indicated by -) + insertion of stop codons in 3 frames.  (mRNA sense).5′ AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCATTGACAGAAGATGGAGAAGGCAAAGCAGAACTAGCAGAAAAATTACACTGTTGGTTCGGTGGGAAAGAATTTGACCTAGACTCTGCCTTGGAATGGATAAAAAACAAAAGATGCTTAACTGACATACAGAAAGCACTAATTGGCGCCTCTATCTGCTTTTTAAAACCCAAAGACCAGGAAAGAAAAAGAAGATTCATCACAGAGCCCCTATCAGGAATGGGGACAACAGCAACAAAAAAGAAGGGCCTGATTCTAGCTGAGAGAAAAATGAGAAGATGTGTGAGCTTCCATGAAGCATTTGAAATAGCAGAAGGCCATGAAAGCTCAGCGTTACTATATTGTCTCATGGTCATGTACCTGAATCCTGGAAATTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTGTGCGAAAAACAAGCATCACATTCACACAGGGCTCATAGCAGAGCAGCGAGATCTTCAGTGCCTGGAGTGAGACGGGAAATGCAGATGGTCTCAGCTATGAACACAGCAAAAACAATGAATGGAATGGGAAAAGGAGAAGACGTTCAAAAACTGGCAGAAGAACTGCAAAGCAACATTGGAGTATTGAGATCTCTTGGGGCAAGTCAAAAGAATGGGGAAGGAATTGCAAAGGATGTAATGGAAGTGCTAAAGCAGAGCTCTATGGGAAATTCAGCTCTTGTGAAGAAATACCTATAATGCTCGAACCATTTCAGATTCTTTCAATTTGTtagAtagC------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------taaATTCAATTTTTACTGTACTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGCAAATAAACTGGAAAAAGTGCGTTGTTTCTACTBOLD UPPER CASE = BM1 ORF Stop Codonbold lower case = Inserted BM2 Stop Codons- = designates deleted nucleotidesBM2SR-3 (SEQ ID NO: 8) influenza B/FL/4/2006Segment 7 with intact BM1 + BM1 M86Vmutation + partial BM2 deletion of 296 bp(indicated by -) + insertion of stop codons in 3 frames. (mRNA sense).5′ AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCATTGACAGAAGATGGAGAAGGCAAAGCAGAACTAGCAGAAAAATTACACTGTTGGTTCGGTGGGAAAGAATTTGACCTAGACTCTGCCTTGGAATGGATAAAAAACAAAAGATGCTTAACTGACATACAGAAAGCACTAATTGGCGCCTCTATCTGCTTTTTAAAACCCAAAGACCAGGAAAGAAAAAGAAGATTCATCACAGAGCCCCTA TCAGGA gTGGGGACAACAGCAACAAAAAAGAAGGGCCTGATTCTAGCTGAGAGAAAAATGAGAAGATGTGTGAGCTTCCATGAAGCATTTGAAATAGCAGAAGGCCATGAAAGCTCAGCGTTACTATATTGTCTCATGGTCATGTACCTGAATCCTGGAAATTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTGTGCGAAAAACAAGCATCACATTCACACAGGGCTCATAGCAGAGCAGCGAGATCTTCAGTGCCTGGAGTGAGACGGGAAATGCAGATGGTCTCAGCTATGAACACAGCAAAAACAATGAATGGAATGGGAAAAGGAGAAGACGTTCAAAAACTGGCAGAAGAACTGCAAAGCAACATTGGAGTATTGAGATCTCTTGGGGCAAGTCAAAAGAATGGGGAAGGAATTGCAAAGGATGTAATGGAAGTGCTAAAGCAGAGCTCTATGGGAAATTCAGCTCTTGTGAAGAAATACCTATA ATGCTCGAA CCATTTCAGATTCTTTCAATTTGTtagAtagC-------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------taaATTCAATTTTTACTGTACTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGCAAATAAACTGGAAAAAGTGCGTTGTTTCTACTBold Underline Mixed Case = BM1 M86V Mutation CodonBOLD UPPER CASE = BM1 ORF Stop CodonUNDERLINE UPPERCASE = BM2 ORF remnantbold lower case = Inserted BM2 Stop Codons- = designates deleted nucleotidesBM2SR-4 (SEQ ID NO: 9) influenza B/FL/4/2006Segment 7 with intact BM1 + partial BM2deletion of 90 bp (indicated by -) + insertionof 3 stop codons in 3 frames. (mRNA sense).5′ AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCATTGACAGAAGATGGAGAAGGCAAAGCAGAACTAGCAGAAAAATTACACTGTTGGTTCGGTGGGAAAGAATTTGACCTAGACTCTGCCTTGGAATGGATAAAAAACAAAAGATGCTTAACTGACATACAGAAAGCACTAATTGGCGCCTCTATCTGCTTTTTAAAACCCAAAGACCAGGAAAGAAAAAGAAGATTCATCACAGAGCCCCTATCAGGAATGGGGACAACAGCAACAAAAAAGAAGGGCCTGATTCTAGCTGAGAGAAAAATGAGAAGATGTGTGAGCTTCCATGAAGCATTTGAAATAGCAGAAGGCCATGAAAGCTCAGCGTTACTATATTGTCTCATGGTCATGTACCTGAATCCTGGAAATTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTGTGCGAAAAACAAGCATCACATTCACACAGGGCTCATAGCAGAGCAGCGAGATCTTCAGTGCCTGGAGTGAGACGGGAAATGCAGATGGTCTCAGCTATGAACACAGCAAAAACAATGAATGGAATGGGAAAAGGAGAAGACGTTCAAAAACTGGCAGAAGAACTGCAAAGCAACATTGGAGTATTGAGATCTCTTGGGGCAAGTCAAAAGAATGGGGAAGGAATTGCAAAGGATGTAATGGAAGTGCTAAAGCAGAGCTCTATGGGAAATTCAGCTCTTGTGAAGAAATACCTATA ATGCTCGAA CCATTTCAGATTCTTTCAATTTGTtagAtagCtaa----------- ----------------------------------------------------------------AAGGGGCCAAATAAAGAGACAATAAACAGAGAGGTATCAATTTTGAGACACAGTTACCAAAAAGAAATCCAGGCCAAAGAAGCAATGAAGGAAGTACTCTCTGACAACATGGAGGTATTGAGTGACCACATAGTAATTGAGGGGCTTTCTGCTGAAGAGATAATAAAAATGGGTGAAACAGTTTTGGAGGTAGAAGAATTGCAT TAA ATTCAATTTTTACTGTACTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGCAAATAAA-CTGGAAAAAGTGCGTTGTTTCTACTBOLD UPPER CASE = BM1 ORF Stop CodonUNDERLINE UPPERCASE = BM2 ORF remnantBOLD UNDERLINE UPPERCASE = BM2 ORF Stop Codonbold lower case = Inserted BM2 Stop Codons- = designates deleted nucleotidesBM2SR-5 (SEQ ID NO: 10) influenza B/FL/4/2006Segment 7 with intact BM1 + BM1 M86Vmutation + partial BM2 deletion of 90 bp(indicated by -) + insertion of 3 stop codons in 3 frames. (mRNA sense).5′ AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCATTGACAGAAGATGGAGAAGGCAAAGCAGAACTAGCAGAAAAATTACACTGTTGGTTCGGTGGGAAAGAATTTGACCTAGACTCTGCCTTGGAATGGATAAAAAACAAAAGATGCTTAACTGACATACAGAAAGCACTAATTGGCGCCTCTATCTGCTTTTTAAAACCCAAAGACCAGGAAAGAAAAAGAAGATTCATCACAGAGCCCCTA TCAGGA gTGGGGACAACAGCAACAAAAAAGAAGGGCCTGATTCTAGCTGAGAGAAAAATGAGAAGATGTGTGAGCTTCCATGAAGCATTTGAAATAGCAGAAGGCCATGAAAGCTCAGCGTTACTATATTGTCTCATGGTCATGTACCTGAATCCTGGAAATTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTGTGCGAAAAACAAGCATCACATTCACACAGGGCTCATAGCAGAGCAGCGAGATCTTCAGTGCCTGGAGTGAGACGGGAAATGCAGATGGTCTCAGCTATGAACACAGCAAAAACAATGAATGGAATGGGAAAAGGAGAAGACGTTCAAAAACTGGCAGAAGAACTGCAAAGCAACATTGGAGTATTGAGATCTCTTGGGGCAAGTCAAAAGAATGGGGAAGGAATTGCAAAGGATGTAATGGAAGTGCTAAAGCAGAGCTCTATGGGAAATTCAGCTCTTGTGAAGAAATACCTATA ATGCTCGAA CCATTTCAGATTCTTTCAATTTGTtagAtagCtaa----------- ----------------------------------------------------------------AAGGGGCCAAATAAAGAGACAATAAACAGAGAGGTATCAATTTTGAGACACAGTTACCAAAAAGAAATCCAGGCCAAAGAAGCAATGAAGGAAGTACTCTCTGACAACATGGAGGTATTGAGTGACCACATAGTAATTGAGGGGCTTTCTGCTGAAGAGATAATAAAAATGGGTGAAACAGTTTTGGAGGTAGAAGAATTGCAT TAA ATTCAATTTTTACTGTACTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGCAAATAAA-CTGGAAAAAGTGCGTTGTTTCTACTBold Underline Mixed Case = BM1 M86V Mutation CodonBOLD UPPER CASE = BM1 ORF Stop CodonUNDERLINE UPPERCASE = BM2 ORF remnantbold lower case = Inserted BM2 Stop CodonsBOLD UNDERLINE UPPERCASE = BM2 ORF Stop Codon- = designates deleted nucleotidesBM2SR-0 (SEQ ID NO: 11)) influenza B/Lee/1940Segment 7 with intact BM1 + total BM2deletion of 329 bp (indicated by -) (mRNA sense).5′ AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCACTAATAGAAGATGGAGAAGGCAAAGCAGAACTAGCTGAAAAATTACACTGTTGGTTCGGTGGGAAAGAATTTGACCTAGATTCTGCTTTGGAATGGATAAAAAACAAAAGGTGCCTAACTGATATACAAAAAGCACTAATTGGTGCCTCTATATGCTTTTTAAAACCCAAAGACCAAGAAAGAAAAAGGAGATTCATCACAGAGCCCCTGTCAGGAATGGGAACAACAGCAACAAAGAAGAAAGGCCTAATTCTAGCTGAGAGAAAAATGAGAAGATGTGTAAGCTTTCATGAAGCATTTGAAATAGCAGAAGGCCACGAAAGCTCAGCATTACTATATTGTCTTATGGTCATGTACCTAAACCCTGAAAACTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTATGCGAGAAACAAGCATCGCACTCGCATAGAGCCCATAGCAGAGCAGCAAGGTCTTCGGTACCTGGAGTAAGACGAGAAATGCAGATGGTTTCAGCTATGAACACAGCAAAGACAATGAATGGAATGGGAAAGGGAGAAGACGTCCAAAAACTAGCAGAAGAGCTGCAAAACAACATTGGAGTGTTGAGATCTCTAGGAGCAAGTCAAAAGAATGGAGAAGGAATTGCCAAAGATGTAATGGAAGTGCTAAAACAGAGCTCTATGGGAAATTCAGCTCTTGTGAGGAAATACTTATAA-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GCCCAATTTTCACTGTATTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGTGAATAAAACTGGAAAAAGTGCGTTGTTTCTACTBOLD UPPER CASE = BM1 ORF Stop Codon - = designates deleted nucleotides

The influenza B genomic segment 7 expresses two major polypeptides thatare required by the virus for replication, the BM1 matrix protein andthe BM2 proton channel. Expression of the BM1 and the BM2 polypeptidesis regulated in part by a pentanucleotide motif translational slippagesite that lies at the junction between the BM1 and BM2 ORFs. Thepentanucleotide motif, TAATG, contains both a TAA stop codon fortermination of M1 translation and an ATG start codon for initiation ofM2 in an alternate-1 reading frame. This pentanucleotide motif andflanking sequences have been shown to be important for the regulation ofexpression of the M1 protein.

The wild-type influenza B segment 7 showing the BM1 and BM2 codingsequences and the pentanucleotide motif in bold underlining are providedbelow in Table 6.

TABLE 6 Wild-type BM1 and BM2 coding sequence(SEQ ID NO: 12) influenza B/Lee/40 Segment 7.AGCAGAAGCACGCACTTTCTTAAAATGTCGCTGTTTGGAGACACAATTGCCTACCTGCTTTCACTAATAGAAGATGGAGAAGGCAAAGCAGAACTAGCTGAAAAATTACACTGTTGGTTCGGTGGGAAAGAATTTGACCTAGATTCTGCTTTGGAATGGATAAAAAACAAAAGGTGCCTAACTGATATACAAAAAGCACTAATTGGTGCCTCTATATGCTTTTTAAAACCCAAAGACCAAGAAAGAAAAAGGAGATTCATCACAGAGCCCCTGTCAGGAATGGGAACAACAGCAACAAAGAAGAAAGGCCTAATTCTAGCTGAGAGAAAAATGAGAAGATGTGTAAGCTTTCATGAAGCATTTGAAATAGCAGAAGGCCACGAAAGCTCAGCATTACTATATTGTCTTATGGTCATGTACCTAAACCCTGAAAACTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTATGCGAGAAACAAGCATCGCACTCGCATAGAGCCCATAGCAGAGCAGCAAGGTCTTCGGTACCTGGAGTAAGACGAGAAATGCAGATGGTTTCAGCTATGAACACAGCAAAGACAATGAATGGAATGGGAAAGGGAGAAGACGTCCAAAAACTAGCAGAAGAGCTGCAAAACAACATTGGAGTGTTGAGATCTCTAGGAGCAAGTCAAAAGAATGGAGAAGGAATTGCCAAAGATGTAATGGAAGTGCTAAAACAGAGCTCTATGGGAAATTCAGCTCTTGTGAGGAAATACTTA TAATG CTCGAACCACTTCAGATTCTTTCAATTTGTTCTTTCATTTTATCAGCTCTCCATTTCATGGCTTGGACAATAGGGCATTTGAATCAAATAAGAAGAGGGGTAAACCTGAAAATACAAATAAGGAATCCAAATAAGGAGGCAATAAACAGAGAGGTGTCAATTCTGAGACACAATTACCAAAAGGAAATCCAAGCCAAAGAAACAATGAAGAAAATACTCTCTGACAACATGGAAGTATTGGGTGACCACATAGTAGTTGAAGGGCTTTCAACTGATGAGATAATAAAAATGGGTGAAACAGTTTTGGAGGTGGAAGAATTGCAATGAGCCCAATTTTCACTGTATTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGTGAATAAAACTGGAAAAAGTGCGTTGTTTCTACT

IV. Cell-Based Virus Production System

A. Producing “First Generation” Mutant Viruses

Mutant virus, such as those carrying mutant M2 nucleic acid, can begenerated by plasmid-based reverse genetics as described by Neumann etal., Generation of influenza A viruses entirely from clone cDNAs, Proc.Natl. Acad. Sci. USA 96:9345-9350 (1999), herein incorporated byreference in its entirety. Mutant virus, such as those carrying mutantBM2 nucleic acid, can be generated by similar means. Briefly, eukaryotichost cells are transfected with one or more plasmids encoding the eightviral RNAs. Each viral RNA sequence is flanked by an RNA polymerase Ipromoter and an RNA polymerase I terminator. Notably, the viral RNAencoding the M2 protein includes the mutant M2 nucleic acid sequence.The host cell is additionally transfected with one or more expressionplasmids encoding the viral proteins (e.g., polymerases, nucleoproteinsand structural proteins), including a wild-type M2 protein. Transfectionof the host cell with the viral RNA plasmids results in the synthesis ofall eight influenza viral RNAs, one of which harbors the mutant M2sequence. The co-transfected viral polymerases and nucleoproteinsassemble the viral RNAs into functional vRNPs that are replicated andtranscribed, ultimately forming infectious influenza virus having amutant M2 nucleic acid sequence, yet having a functional M2 polypeptideincorporated into the viral lipid envelope.

Alternative methods of producing a “first generation” mutant virusinclude a ribonucleoprotein (RNP) transfection system that allows thereplacement of influenza virus genes with in vitro generated recombinantRNA molecules, as described by Enami and Palese, High-efficiencyformation of influenza virus transfectants, J. Virol. 65(5):2711-2713,which is incorporated herein by reference.

The viral RNA is synthesized in vitro and the RNA transcripts are coatedwith viral nucleoprotein (NP) and polymerase proteins that act asbiologically active RNPs in the transfected cell as demonstrated byLuytjes et al., Amplification, expression, and packaging of a foreigngene by influenza virus, Cell 59:1107-1113, which is incorporated hereinby reference.

The RNP transfection method can be divided into four steps: 1)Preparation of RNA: plasmid DNA coding for an influenza virus segment istranscribed into negative-sense RNA in an in vitro transcriptionreaction; 2) Encapsidation of the RNA: the transcribed RNA is then mixedwith gradient purified NP and polymerase proteins isolated fromdisrupted influenza virus to form a biologically active RNP complex; 3)Transfection and rescue of the encapsidated RNA: the artificialribonucleocapsid is transfected to the cells previously infected with ahelper influenza virus that contains a different gene from the one beingrescued; the helper virus will amplify the transfected RNA; 4) Selectionof transfected gene: because both the helper virus and the transfectantcontaining the rescued gene are in the culture supernatant, anappropriate selection system using antibodies is necessary to isolatethe virus bearing the transfected gene.

The selection system allows for the generation of novel transfectantinfluenza viruses with specific biological and molecularcharacteristics. Antibody selection against a target surface protein canthen be used for positive or negative selection.

For example, a transfectant or mutant virus that contains an M2 genethat does not express an M2 protein can be grown in a suitable mammaliancell line that has been modified to stably express the wild-typefunctional M2 protein. To prevent or inhibit replication of the helpervirus expressing the wild-type M2 gene, and therefore the M2e protein atthe membrane surface, antibodies against M2e can be used. Suchantibodies are commercially available and would inhibit the replicationof the helper virus and allow for the transfectant/mutant viruscontaining the mutant M2 to grow and be enriched in the supernatant.Inhibition of influenza virus replication by M2e antibodies has beendescribed previously in Influenza A virus M2 protein: monoclonalantibody restriction of virus growth and detection of M2 in virions, JVirol 62:2762-2772 (1988) and Treanor et al, Passively transferredmonoclonal antibody to the M2 protein inhibits influenza A virusreplication in mice, J. Virol. 64:1375-1377 (1990).

Additionally or alternatively, the same antibodies can be used to‘capture’ the helper virus and allow for the enrichment of thetransfectant. For example, the antibodies can be used to coat the bottomof a tissue culture dish or can be used in a column matrix to allow forenrichment for the transfectant in the supernatant or eluate.

The transfectant virus can be grown in M2 expressing cells in multi-wellplates by limit dilution and then be identified and cloned, for example,by creating replica plates. For example, one-half of an aliquot of agiven well of the multi-well plate containing the grown virus can beused to infect MDCK cells and the other half to infect MDCK cells thatexpress M2 protein. Both the transfectant virus and helper virus willgrow in MDCK cells that express M2 protein. However, only helper viruswill grow in standard MDCK cells allowing for identifying the well inthe multi-well plate that contains the transfectant. The transfectantvirus can be further plaque purified in the cells that express M2protein.

B. Propagating Viral Mutants

In some embodiments, viral mutants described herein are maintained andpassaged in host cells. By way of example, but not by way of limitation,exemplary host cells appropriate for growth of influenza viral mutants,such as influenza A viral mutants include any number of eukaryoticcells, including, but not limited to Madin-Darby canine kidney (MDCK)cells, simian cells such as African green monkey cells (e.g., Verocells), CV-1 cells and rhesus monkey kidney cells (e.g., LLcomk.2cells), bovine cells (e.g., MDBK cells), swine cells, ferret cells(e.g., mink lung cells) BK-1 cells, rodent cells (e.g., Chinese HamsterOvary cells), human cells, e.g., embryonic human retinal cells (e.g.,PER-C6®), 293T human embryonic kidney cells and avian cells includingembryonic fibroblasts.

Additionally or alternatively, in some embodiments, the eukaryotic hostcell is modified to enhance viral production, e.g., by enhancing viralinfection of the host cell and/or by enhancing viral growth rate. Forexample, in some embodiments, the host cell is modified to express, orto have increased expression, of 2,6-linked sialic acid on the cellsurface, allowing for more efficient and effective infection of thesecells by mutant or wild-type influenza A viruses. See e.g., U.S. PatentPublication No. 2010-0021499, and U.S. Pat. No. 7,176,021, hereinincorporated by reference in their entirety. Thus, in some illustrativeembodiments, Chinese Hamster Ovary Cells (CHO cells) and/or Vero cellsmodified to express at least one copy of a 2,6-sialyltransferase gene(ST6GAL 1) are used. By way of example, but not by way of limitation,the Homo sapiens ST6 beta-galatosamide alpha-2,6-sialyltransferase genesequence denoted by the accession number BC040009.1, is one example of aST6Gal gene that can be integrated into and expressed by a CHO cell. Oneor more copies of a polynucleotide that encodes a functional ST6Gal Igene product can be engineered into a cell. That is, cells which havebeen stably transformed to express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or more than 12 copies of a ST6Gal I gene may be used. A singleexpression cassette may include one or more copies of the ST6Gal I geneto be expressed, which is operably linked to regulatory elements, suchas promoters, enhancers, and terminator and polyadenylation signalsequences, to facilitate the expression of the ST6Gal I gene or itscopies. Alternatively, a single expression cassette may be engineered toexpress one copy of an ST6Gal I gene, and multiple expression cassettesintegrated into a host cell genome. Accordingly, in some embodiments, atleast one ST6Gal I gene is incorporated into the genome of a host cell,such that the cell expresses the ST6Gal I gene and its enzymatic proteinproduct. Depending on the copy number, a single host cell may expressmany functional ST6Gal I gene proteins.

Suitable vectors for cloning, transfecting and producing stable,modified cell lines are well known in the art. One non-limiting exampleincludes the pcDNA3.1 vectors (Invitrogen).

Additionally or alternatively, in some embodiments, the eukaryotic hostcell is modified to produce a wild-type version of a mutant viral gene,thereby providing the gene to the virus in trans. For example, a viralstrain harboring a mutant M2 protein may exhibit an enhanced growth rate(e.g., greater viral production) when passaged in host cells producingthe wild-type M2 protein. In some embodiments, the a viral strainharboring a mutant M2 protein may not grow or replicate in a cell whichdoes not express a wild-type M2 gene. In addition, such host cells mayslow or prevent viral reversion to a functional M2 sequence, because,for example, there is no selective pressure for reversion in such ahost.

Methods for producing both expression vectors and modified host cellsare well known in the art. For example, an M2 expression vector can bemade by positioning the M2 nucleic acid sequence (M2 ORF sequence; thisis “wild-type” M2's start codon to stop codon (Table 7)) below in aeukaryotic expression vector. Similar methods can be employed for BM2,the sequence of which is provided below in Table 7.

TABLE 7 Wild-type M2 nucleic acid sequence (SEQ ID NO: 13)ATGAGTCTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGGTGCAGATGCAACGGTTCAAGTGATCCTCTCACTATTGCCGCAAATATCATTGGGATCTTGCACTTGACATTGTGGATTCTTGATCGTCTTTTTTTCAAATGCATTTACCGTCGCTTTAAATACGGACTGAAAGGAGGGCCTTCTACGGAAGGAGTGCCAAAGTCTATGAGGGAAGAATATCGAAAGGAACAGCAGAGTGCTGTGGATGCTGACGATGGTCATTTTGTCAGCATAGAGCTG GAGTAA

TABLE 8 Wild-type BM2 nucleic acid sequence (SEQ ID NO: 14)ATGCTCGAACCACTTCAGATTCTTTCAATTTGTTCTTTCATTTTATCAGCTCTCCATTTCATGGCTTGGACAATAGGGCATTTGAATCAAATAAGAAGAGGGGTAAACcTGAAAATACAAATAAGGAATCCAAATAAGGAGGCAATAAACAGAGAGGTGTCAATTCTGAGACACAATTACCAAAAGGAAATCCAAGCCAAAGAAACAATGAAGAAAATACTCTCTGACAACATGGAAGTATTGGGTGACCACATAGTAGTTGAAGGGCTTTCAACTGATGAGATAATAAAAATGGGTGAAACAGTTTTGGAGGTGGAAGAATTGCAATGAGCCCAATTTTCACTGTATTTCTTACTATGCATTTAAGCAAATTGTAATCAATGTCAGTGAATAAAACTGGAAAAAGTGCGTTGT TTCTACT

Host cells (e.g., MDCK cells) can then be transfected by methods knownin the art, e.g., using commercially available reagents and kits, suchas TransIT® LT1 (Minis Bio, Madison, Wis.). By way of example, but notby way of limitation, cells can be selected and tested for M2 expressionby cotransfection with a detectable marker or a selectable marker (e.g.,hygromycin-resistance) and/or by screening, for example, with indirectimmunostaining using an M2 antibody. M2 expression can be determined byindirect immunostaining, flow cytometry or ELISA.

By way of example, but not by way of limitation, 293T human embryonickidney cells and Madin-Darby canine kidney (MDCK) cells were maintainedin Dulbecco's modified Eagle's medium supplemented with 10% fetal calfserum and in minimal essential medium (MEM) containing 5% newborn calfserum, respectively. All cells were maintained at 37° C. in 5% CO₂.Hygromycin-resistant MDCK cells stably expressing M2 protein fromA/Puerto Rico/8/34 (H1N1) were established by cotransfection withplasmid pRHyg, containing the hygromycin resistance gene, and plasmidpCAGGS/M2, expressing the full-length M2 protein, at a ratio of 1:1. Thestable MDCK cell clone (M2CK) expressing M2 was selected in mediumcontaining 0.15 mg/mL of hygromycin (Roche, Mannheim, Germany) byscreening with indirect immunostaining using an anti-M2 (14C2)monoclonal antibody (Iwatsuki et al., JVI, 2006, vol. 80, No. 1, p.5233-5240). The M2CK cells were cultured in MEM supplemented with 10%fetal calf serum and 0.15 mg/mL of hygromycin. In M2CK cells, theexpression levels and localization of M2 were similar to those invirus-infected cells (data not shown). BM2-expressing BM2CK cells can bemade in a similar fashion, and M2- or BM2-expressing Vero cells can bemade in a similar fashion.

In some embodiments, cells and viral mutants are cultured and propagatedby methods well known in the art. By way of example, but not by way oflimitation, in some embodiments, host cells are grown in the presence ofMEM supplemented with 10% fetal calf serum. Cells expressing M2 or BM2are infected at an MOI of 0.001 by washing with PBS followed byadsorbing virus at 37° C. In some embodiments, viral growth mediacontaining trypsin/TPCK is added and the cells are incubated for 2-3days until cytopathic effect is observed.

Along these lines, disposable bioreactor systems have been developed formammalian cells, with or without virus, whose benefits include fasterfacility setup and reduced risk of cross-contamination. The cellsdescribed herein, for instance, can be cultured in disposable bags suchas those from Stedim, Bioeaze bags from SAFC Biosciences, HybridBag™from Cellexus Biosytems, or single use bioreactors from HyClone orCelltainer from Lonza. Bioreactors can be 1 L, 10 L, 50 L, 250 L, 1000 Lsize formats. In some embodiments, the cells are maintained insuspension in optimized serum free medium, free of animal products. Thesystem can be a fed-batch system where a culture can be expanded in asingle bag from 1 L to 10 L for example, or a perfusion system thatallows for the constant supply of nutrients while simultaneouslyavoiding the accumulation of potentially toxic by-products in theculture medium.

For long term storage, mutant virus can be stored as frozen stocks.

V. Vaccines and Method of Administration

A. Immunogenic Compositions and Vaccines

There are various different types of vaccines which can be made from thecell-based virus production system disclosed herein. The presentdisclosure includes, but is not limited to, the manufacture andproduction of live attenuated virus vaccines, single replicationvaccines, replication defective vaccines, viral vector vaccines,inactivated virus vaccines, whole virus vaccines, split virus vaccines,virosomal virus vaccines, viral surface antigen vaccines andcombinations thereof. Thus, there are numerous vaccines capable ofproducing a protective immune response specific for different influenzaviruses where appropriate formulations of any of these vaccine types arecapable of producing an immune response, e.g., a systemic immuneresponse. Live attenuated virus vaccines have the advantage of alsobeing able to stimulate local mucosal immunity in the respiratory tract.

In some embodiments, vaccine antigens used in the compositions describedherein are “direct” antigens, i.e., they are not administered as DNA,but are the antigens themselves. Such vaccines may include a whole virusor only part of the virus, such as, but not limited to viralpolysaccharides, whether they are alone or conjugated to carrierelements, such as carrier proteins, live attenuated wholemicroorganisms, inactivated microorganisms, recombinant peptides andproteins, glycoproteins, glycolipids, lipopeptides, synthetic peptides,or ruptured microorganisms in the case of vaccines referred to as“split” vaccines.

In some embodiments, a complete virion vaccine is provided. A completevirion vaccine can be concentrated by ultrafiltration and then purifiedby zonal centrifugation or by chromatography. Typically, the virion isinactivated before or after purification using formalin orbeta-propiolactone, for instance.

In some embodiments, a subunit vaccine is provided, which comprisespurified glycoproteins. Such a vaccine may be prepared as follows: usingviral suspensions fragmented by treatment with detergent, the surfaceantigens are purified, by ultracentrifugation for example. The subunitvaccines thus contain mainly HA protein, and also NA. The detergent usedmay be cationic detergent for example, such as hexadecyl trimethylammonium bromide, an anionic detergent such as ammonium deoxycholate; ora nonionic detergent such as that commercialized under the name TRITONX100. The hemagglutinin may also be isolated after treatment of thevirions with a protease such as bromelin, then purified by standardmethods.

In some embodiments, a split vaccine is provided, which comprisesvirions which have been subjected to treatment with agents that dissolvelipids. A split vaccine can be prepared as follows: an aqueoussuspension of the purified virus obtained as above, inactivated or not,is treated, under stirring, by lipid solvents such as ethyl ether orchloroform, associated with detergents. The dissolution of the viralenvelope lipids results in fragmentation of the viral particles. Theaqueous phase is recuperated containing the split vaccine, constitutedmainly of hemagglutinin and neuraminidase with their original lipidenvironment removed, and the core or its degradation products. Then theresidual infectious particles are inactivated if this has not alreadybeen done.

In some embodiments, inactivated influenza virus vaccines are provided.In some embodiments, the inactivated vaccines are made by inactivatingthe virus using known methods, such as, but not limited to, formalin orβ-propiolactone treatment. Inactivated vaccine types that can be used inthe invention can include whole-virus (WV) vaccines or subvirion (SV)(split) vaccines. The WV vaccine contains intact, inactivated virus,while the SV vaccine contains purified virus disrupted with detergentsthat solubilize the lipid-containing viral envelope, followed bychemical inactivation of residual virus.

Additionally or alternatively, in some embodiments, live attenuatedinfluenza virus vaccines are provided. Such vaccines can be used forpreventing or treating influenza virus infection, according to knownmethod steps.

In some embodiments, attenuation is achieved in a single step bytransfer of attenuated genes from an attenuated donor virus to anisolate or reassorted virus according to known methods (see, e.g.,Murphy, Infect. Dis. Clin. Pract. 2, 174 (1993)). In some embodiments, avirus is attenuated by mutation of one or more viral nucleic acidsequences, resulting in a mutant virus. For example, in someembodiments, the mutant viral nucleic acid sequence codes for adefective protein product. In some embodiments, the protein product hasdiminished function or no function. In other embodiments, no proteinproduct is produced from the mutant viral nucleic acid.

The single replication virus described herein can be formulated andadministered according to known methods, as an immunogenic composition(e.g., as a vaccine) to induce an immune response in an animal, e.g., anavian and/or a mammal. Methods are well-known in the art for determiningwhether such attenuated or inactivated vaccines have maintained similarantigenicity to that of the clinical isolate or a high growth strainderived therefrom. Such known methods include the use of antisera orantibodies to eliminate viruses expressing antigenic determinants of thedonor virus; chemical selection (e.g., amantadine or rimantidine); HAand NA activity and inhibition; and DNA screening (such as probehybridization or PCR) to confirm that donor genes encoding the antigenicdeterminants (e.g., HA or NA genes) or other mutant sequences (e.g., M2)are not present in the attenuated viruses. See, e.g., Robertson et al.,Giornale di Igiene e Medicina Preventiva, 29, 4 (1988); Kilbourne, Bull.M2 World Health Org., 41, 643 (1969); and Robertson et al., Biologicals,20, 213 (1992).

In some embodiments, the vaccine includes a single replication influenzavirus that lacks expression of a functional M2 protein. In someembodiments, the mutant virus replicates well in cells expressing M2proteins, but in the corresponding wild-type cells, expresses viralproteins without generating infectious progeny virions.

Pharmaceutical compositions of the present technology, suitable forintranasal administration, intradermal administration, inoculation, orfor parenteral or oral administration, comprise attenuated orinactivated influenza viruses, and may optionally further comprisesterile aqueous or non-aqueous solutions, suspensions, and emulsions.The compositions can further comprise auxiliary agents or excipients, asknown in the art. See, e.g., Berkow et al., The Merck Manual, 15thedition, Merck and Co., Rahway, N.J. (1987); Goodman et al., eds.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, EighthEdition, Pergamon Press, Inc., Elmsford, N.Y. (1990); Avery's DrugTreatment: Principles and Practice of Clinical Pharmacology andTherapeutics, Third Edition, ADIS Press, LTD., Williams and Wilkins,Baltimore, Md. (1987); and Katzung, ed., Basic and ClinicalPharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn. (1992).

In some embodiments, liquid preparations for intranasal delivery maytake the form of solutions or suspensions and may contain conventionalexcipients such as tonicity adjusting agents, for example, sodiumchloride, dextrose or mannitol; preservatives, for example benzalkoniumchloride, thiomersal, phenylethyl alcohol; and other formulating agentssuch as suspending, buffering, stabilising and/or dispersing agents.

In some embodiments, preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and/or emulsions,which may contain auxiliary agents or excipients known in the art.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers or occlusive dressings can be used toincrease skin permeability and enhance antigen absorption. Liquid dosageforms for oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

When a composition of the present invention is used for administrationto an individual, it can further comprise salts, buffers, adjuvants, orother substances which are desirable for improving the efficacy of thecomposition. For vaccines, adjuvants (substances that augment a specificimmune response) can be used. Normally, the adjuvant and the compositionare mixed prior to presentation to the immune system, or presentedseparately, but into the same site of the organism being immunized.

In some embodiments, the present disclosure provides a multivalentimmunogenic composition comprising viruses from at least two influenzastrains. In some embodiments, the multivalent immunogenic compositioncomprises: (a) at least one engineered attenuated influenza AM2-deficient recombinant virus, wherein the engineered influenza Aviruses comprise a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2,or SEQ ID NO: 3; and (b) at least one engineered attenuated influenzaBM2-deficient recombinant virus, wherein the engineered influenza Bvirus comprises a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

In some embodiments, the influenza A viruses are chosen from the groupof H1N1 and H3N2 subtypes, and the influenza B viruses are chosen fromthe group of B/Yamagata and B/Victoria lineages.

In some embodiments, the present disclosure provides a quadrivalentimmunogenic composition comprising: two M2-deficient influenza A M2SRviruses: A/California/07/2009 (H1N1) and A/Brisbane/10/2007 (H3N2), bothof which comprise an M2SR-1 mutant comprising SEQ ID NO: 1; and twoBM2-deficient influenza B BM2SR viruses: B/Brisbane/60/2008 (Victoria)and B/Wisconsin/01/2010 (Yamagata) both of which comprise a BM2SR-0mutant comprising SEQ ID NO: 11. In some embodiments, this immunogeniccomposition is formulated as a quadrivalent influenza vaccine.

In some embodiments, the present disclosure provides a method ofstimulating an immune response against influenza A and influenza B,comprising administering to a subject in need thereof a multivalentimmunogenic composition comprising from at least one influenza A strainand at least one influenza B strain. In some embodiments, themultivalent immunogenic composition comprises: (a) at least oneengineered attenuated influenza A M2-deficient recombinant virus,wherein the engineered influenza A viruses comprise a mutant M2 genecomprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and (b) at leastone engineered attenuated influenza BM2-deficient recombinant virus,wherein the engineered influenza B virus comprises a mutant BM2 genecomprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, or SEQ ID NO: 11.

In some embodiments, the influenza A viruses are chosen from the groupof H1N1 and H3N2 subtypes, and the influenza B viruses are chosen fromthe group of B/Yamagata and B/Victoria lineages.

In some embodiments, the present disclosure provides a method ofstimulating an immune response against influenza A and influenza B,comprising administering to a subject in need thereof an immunogeniccomposition comprising: two M2-deficient influenza A M2SR viruses:A/California/07/2009 (H1N1) comprising an M2SR-1 mutant comprising SEQID NO: 1 and A/Brisbane/10/2007 (H3N2) comprising an M2SR-1 mutantcomprising SEQ ID NO: 1; and two BM2-deficient influenza B BM2SRviruses: B/Brisbane/60/2008 (Victoria) comprising a BM2SR-0 mutantcomprising SEQ ID NO: 11 and B/Wisconsin/01/2010 (Yamagata) comprising aBM2SR-0 mutant comprising SEQ ID NO: 11. In some embodiments, thisimmunogenic composition is formulated as a quadrivalent influenzavaccine.

In some embodiments, the immunogenic composition formulated as aquadrivalent influenza vaccine as described herein exhibits attenuatedvirulence. For example, in some embodiments, mice infected with thequadrivalent vaccine have an increased average post-infection lifespanafter influenza A challenge compared to mice infected with influenza Bmonovalent vaccines alone. In some embodiments, mice infected with thequadrivalent vaccine have an increased average post-infection lifespanfollowing influenza B challenge compared to mice infected with influenzaA monovalent vaccines alone.

A pharmaceutical composition according to the present invention mayfurther or additionally comprise at least one chemotherapeutic compound,e.g., for gene therapy, an immunosuppressant, an anti-inflammatory agentor an immunostimulatory agent, or anti-viral agents including, but notlimited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole,interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α,thiosemicarbarzones, methisazone, rifampin, ribavirin, a pyrimidineanalog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir,dideoxynucleosides, a protease inhibitor, or ganciclovir.

The composition can also contain variable but small quantities ofendotoxin-free formaldehyde, and preservatives, which have been foundsafe and not contributing to undesirable effects in the organism towhich the composition of the invention is administered.

B. Administration

An immunogenic composition (e.g., vaccine) as disclosed herein may beadministered via any of the routes conventionally used or recommendedfor vaccines: parenteral route, mucosal route, and may be in variousforms: injectable or sprayable liquid, formulation which has beenfreeze-dried or dried by atomization or air-dried, etc. Vaccines may beadministered by means of a syringe or by means of a needle-free injectorfor intramuscular, subcutaneous or intradermal injection. Vaccines mayalso be administered by means of a nebulizer capable of delivering a drypowder or a liquid spray to the mucous membranes, whether they arenasal, pulmonary, vaginal or rectal.

A vaccine as disclosed herein may confer resistance to one or moreinfluenza strains by either passive immunization or active immunization.In active immunization, an inactivated or attenuated live vaccinecomposition is administered prophylactically to a host (e.g., a mammal),and the host's immune response to the administration protects againstinfection and/or disease. For passive immunization, the elicitedantisera can be recovered and administered to a recipient suspected ofhaving an infection caused by at least one influenza virus strain.

The present invention thus includes methods for preventing orattenuating a disease or disorder, e.g., infection by at least oneinfluenza virus strain. As used herein, a vaccine is said to prevent orattenuate a disease if its administration results either in the total orpartial attenuation (i.e., suppression) of a symptom or condition of thedisease, or in the total or partial immunity of the individual to thedisease.

At least one inactivated or attenuated influenza virus, or compositionthereof, of the present invention may be administered by any means thatachieve the intended purposes, using a pharmaceutical composition aspreviously described. For example, administration of such a compositionmay be by various parenteral routes such as subcutaneous, intravenous,intradermal, intramuscular, intraperitoneal, intranasal, oral ortransdermal routes. Parenteral administration can be by bolus injectionor by gradual perfusion over time. In some embodiments, an immunogeniccomposition as disclosed herein is by intramuscular or subcutaneousapplication.

In some embodiments, a regimen for preventing, suppressing, or treatingan influenza virus related pathology comprises administration of aneffective amount of a vaccine composition as described herein,administered as a single treatment, or repeated as enhancing or boosterdosages, over a period up to and including between one week and about 24months, or any range or value therein. In some embodiments, an influenzavaccine as disclosed herein is administered annually.

According to the present technology, an “effective amount” of a vaccinecomposition is one that is sufficient to achieve a desired biologicaleffect. It is understood that, in some embodiments, the effective dosagewill be dependent upon the age, sex, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect wanted. The ranges of effective dosesprovided below are not intended to be limiting and represent exemplarydose ranges. Thus, in some embodiments, the dosage will be tailored tothe individual subject, as is understood and determinable by one ofskill in the art. The dosage of an attenuated virus vaccine for amammalian (e.g., human) adult can be from about 10¹-10¹⁰ plaque formingunits (PFU)/kg, or any range or value therein. In some embodiments, thedosage of an attenuated virus vaccine for a mammalian (e.g., human)adult can be from about 10²-10¹⁰ plaque forming units (PFU)/kg, or anyrange or value therein. In some embodiments, the dosage of an attenuatedvirus vaccine for a mammalian (e.g., human) adult can be from about10³-10¹⁰ plaque forming units (PFU)/kg, or any range or value therein.In some embodiments, the dosage of an attenuated virus vaccine for amammalian (e.g., human) adult can be from about 10⁴-10¹⁰ plaque formingunits (PFU)/kg, or any range or value therein. In some embodiments, thedosage of an attenuated virus vaccine for a mammalian (e.g., human)adult can be from about 10⁵-10¹⁰ plaque forming units (PFU)/kg, or anyrange or value therein. In some embodiments, the dosage of an attenuatedvirus vaccine for a mammalian (e.g., human) adult can be from about10⁶-10¹⁰ plaque forming units (PFU)/kg, or any range or value therein.In some embodiments, the dosage of an attenuated virus vaccine for amammalian (e.g., human) adult can be from about 10⁷-10¹⁰ plaque formingunits (PFU)/kg, or any range or value therein. In some embodiments, thedosage of an attenuated virus vaccine for a mammalian (e.g., human)adult can be from about 10⁸-10¹⁰ plaque forming units (PFU)/kg, or anyrange or value therein. In some embodiments, the dosage of an attenuatedvirus vaccine for a mammalian (e.g., human) adult can be from about10⁹-10¹⁰ plaque forming units (PFU)/kg, or any range or value therein.In some embodiments, the dosage of an attenuated virus vaccine for amammalian (e.g., human) adult can be from about 10⁹-10¹⁰ plaque formingunits (PFU)/kg, or any range or value therein. In some embodiments, thedosage of an attenuated virus vaccine for a mammalian (e.g., human)adult can be greater than 10¹⁰ plaque forming units (PFU)/kg. The doseof inactivated vaccine can range from about 0.1 to 200, e.g., 50 μg ofhemagglutinin protein. However, the dosage should be a safe andeffective amount as determined by conventional methods, using existingvaccines as a starting point.

C. Intracutaneous Delivery

Live flu vaccines are traditionally delivered intranasally to mimic thenatural route of infection and promote a similar immune response to thatof natural virus infection. As an alternative, disclosed herein areintradermal delivery methods which involve the use of a novelmicroneedle device to capitalize on the immunological benefits ofintradermal delivery. In some embodiments, an attenuated virus (e.g., anM2 and/or BM2 viral mutant) is used in a vaccine composition forintradermal administration. In some embodiments, M2 and BM2 viralmutants, which do not produce infectious progeny virus, are provided ina quadrivalent vaccine. Thus, any potential of reassortment withwild-type circulating influenza viruses is virtually eliminated.

In embodiments disclosed herein, intradermal delivery (intracutaneous)administers vaccine to the skin. In some embodiments, intradermaldelivery is performed using a microneedle delivery device. As disclosedherein, intracutaneous delivery has numerous advantages. For example,the immunogenicity of the vaccine is enhanced by triggering theimmunological potential of the skin immune system. The vaccine hasdirect access to the potent antigen-presenting dendritic cells of theskin, i.e., epidermal Langerhans Cells and dermal dendritic cells. Skincells produce proinflammatory signals which enhance the immune responseto antigens introduced through the skin. Further, the skin immune systemproduces antigen-specific antibody and cellular immune responses.Intradermal delivery allows for vaccine dose sparing, i.e., lower dosesof antigen may be effective, in light of the above factors, whendelivered intracutaneously.

And, because the vaccine is delivered to the skin through the device'smicroneedle array, the risk of unintended needle-sticks is reduced, andintracutaneous vaccine delivery via microneedle array is relativelypainless compared to intramuscular injections with conventional needleand syringe.

Microneedle devices are known in the art, are known in the art,including, for example, those described in published U.S. patentapplications 2012/0109066, 2011/0172645, 2011/0172639, 2011/0172638,2011/0172637, and 2011/0172609. Microneedle devices may be made, forexample, by fabrication from stainless steel sheets (e.g., Trinity BrandIndustries, Georgia; SS 304; 50 μm thick) by wet etching. In someembodiments, individual microneedles have a length of between about 500μm and 1000 μm, e.g., about 750 μm, and a width of between about 100 μmto 500 μm, e.g., about 200 μm. Vaccine can then be applied to themicroneedles as a coating. By way of example, but not by way oflimitation, a coating solution may include 1% (w/v) carboxymethylcellulose sodium salt (low viscosity, USP grad; Carbo-Mer, San DiegoCalif.), 0.5% (w/v) Lutrol F-68 NF (BASF, Mt. Olive, N.J.) and theantigen (e.g., soluble HA protein at 5 ng/ml; live, attenuated virussuch as the M2 and BM2 mutant virus described herein, etc.). To reach ahigher vaccine concentration, the coating solution may be evaporated for5 to 10 minutes at room temperature (˜23° C.). Coating may be performedby a dip coating process. The amount of vaccine per row of microneedlescan be determined by submerging the microneedles into 200 μl ofphosphate-buffered saline (PBS) for 5 minutes and assaying for theantigen by methods known in the art.

In some embodiments, a microneedle device is used that is made mainly ofpolypropylene and stainless steel first-cut pieces that fit togetherwith simple snap fits and heat seals. In some embodiments, the device iscompletely self-contained and includes the vaccine, a pump mechanism, anactivation mechanism, and a microneedle unit. These components arehidden within a plastic cover. With the device, vaccine infusion isinitiated by pressing an actuation button. Pressing the buttonsimultaneously inserts the microneedles into the skin and initiates thepumping mechanism that exerts pressure on the primary drug container.When a spring mechanism exerts sufficient pressure on the vaccinereservoir, vaccine begins to flow through the microneedle array, andinto the skin. In some embodiments, the delivery of the vaccine dose iscompleted within about 2 minutes after actuation of the device. Afterinfusion is complete, the device is gently removed from the skin.

In some embodiments, a method for intracutaneous administration of animmunogenic composition (e.g., quadrivalent vaccine) is provided using amicroneedle device. In some embodiments, the microneedle devicecomprises a puncture mechanism and an immunogenic composition layercomprising a plurality of microneedles capable of puncturing skin andallowing an immunogenic composition to be administered intracutaneously.In some embodiments, the method comprises depressing the puncturemechanism. In some embodiments, the immunogenic composition (e.g.,quadrivalent vaccine) comprises a virus comprising a nucleic acidsequence encoding a mutant M2 and BM2 protein that is expressed or amutant M2 and BM2 protein that is not expressed; wherein the expressedmutant M2 protein comprises, or consists of, the amino acid sequenceencoded by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the BM2protein comprises, or consists of, the amino acid sequence encoded bySEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,or SEQ ID NO: 11. In some embodiments, the microneedle array isinitially positioned inside of a device housing, and upon actuation of alever allows the microneedles to extend through the device bottom andinsert into the skin thereby allowing infusion of the vaccine fluid intothe skin.

The delivery device described herein may be utilized to deliver anysubstance that may be desired. In one embodiment, the substance to bedelivered is a drug, and the delivery device is a drug delivery deviceconfigured to deliver the drug to a subject. As used herein the term“drug” is intended to include any substance delivered to a subject forany therapeutic, preventative or medicinal purpose (e.g., vaccines,pharmaceuticals, nutrients, nutraceuticals, etc.). In one suchembodiment, the drug delivery device is a vaccine delivery deviceconfigured to deliver a dose of vaccine to a subject. In one embodiment,the delivery device is configured to deliver a flu vaccine. Theembodiments discussed herein relate primarily to a device configured todeliver a substance transcutaneously. In some embodiments, the devicemay be configured to deliver a substance directly to an organ other thanthe skin.

EXAMPLES

As described above, the present application provides a novelquadrivalent immunogenic composition comprising influenza A andinfluenza B mutant strains useful in eliciting an immune response in amammal against influenza A and influenza B. The following examples arepresented to illustrate methods of eliciting an immune response with themutants formulated as multivalent vaccines, and methods of testing theattenuated virulence of the multivalent formulations.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results. The examples should in noway be construed as limiting the scope of the present technology, asdefined by the appended claims.

Example 1 BM2SR Mutants Elicit Antibody Responses Against Influenza BVirus Formulated as Quadrivalent Vaccine

An experiment was performed to demonstrate that BM2SR mutant viruseselicit antibody responses when formulated as a quadrivalent vaccine. Thefollowing four monovalent vaccines were formulated together:A/California/07/2009 (H1N1) comprising an M2SR-1 mutant comprising SEQID NO: 1, A/Brisbane/10/2007 (H3N2) comprising an M2SR-1 mutantcomprising SEQ ID NO: 1, B/Brisbane/60/2008 (Victoria) comprising aBM2SR-0 mutant comprising SEQ ID NO: 11, and B/Wisconsin/01/2010(Yamagata) comprising a BM2SR-0 mutant comprising SEQ ID NO: 11. 1×10⁶TCID₅₀ of each monovalent is mixed together such that each quadrivalentdose is 4×10⁶ TCID₅₀ per mouse. The sequence of each of the M2SR-1 andBM2SR-0 mutant constructs is provided in Tables 1 and 5.

Six-week-old BALB/c female mice were inoculated intranasally with thequadrivalent formulation at a dose of 4×10⁶ TCID₅₀ per mouse. A controlgroup of mice was given PBS. Serum samples were taken on days 7, 14, and21 after prime inoculation and on days 35, 42 and 49 after the secondimmunization on day 28. Anti-HA IgG antibody titers from the serumsamples were determined by enzyme-linked immunosorbent assay (ELISA)against B/Wisconsin/01/2010 and B/Brisbane/60/2008. The humoral responseis shown in FIG. 2, which shows that the quadrivalent M2SR and BM2SRvaccine elevated anti-influenza virus antibodies higher than the controlPBS group against both influenza B antigens representing the twoinfluenza B lineages (B/Bris/60 and B/Wisc/01). Mice boosted byquadrivalent vaccine had higher level of anti-influenza HA antibodiesafter the second immunization than after the prime dose.

These results demonstrate that each monovalent BM2SR vaccine is capableof eliciting antigen specific responses in a quadrivalent formulation.

Example 2 M2SR and BM2SR Mutants Elicit Antibody Responses AgainstInfluenza A and Influenza B Viruses Formulated in Multivalent Vaccines

A. BM2SR Mutants Elicit Antibody Responses Against Influenza B VirusFormulated in Multivalent Vaccines

An experiment was performed to demonstrate that BM2SR mutant viruseselicit antibody responses when formulated as a monovalent, bivalent,trivalent, or quadrivalent vaccine with the influenza A H1N1 or H3N2M2SR vaccines. Table A shows how the different formulations of thefollowing four monovalent M2SR and BM2SR vaccines were formulatedtogether: A/California/07/2009 (H1N1) (comprising an M2SR-1 mutantcomprising SEQ ID NO: 1, A/Brisbane/10/2007 (H3N2) (comprising an M2SR-1mutant comprising SEQ ID NO: 1), B/Brisbane/60/2008 (Victoria)(comprising a BM2SR-0 mutant comprising SEQ ID NO: 11),B/Wisconsin/01/2010 (Yamagata) (comprising a BM2SR-0 mutant comprisingSEQ ID NO: 11).

TABLE A Multivalent formulations of M2SR and BM2SR Mouse Grouping (N =5) Total M2SR viruses virus (1 × 10{circumflex over ( )}6 TCID50/mouseeach) titer A/ A/ B/ B/ (TCID50) CA07 Bris10 WI01 Bris60 per groups(H1N1) (H3N2) (Yamagata) (Victoria) mouse 1 monovalent X 1 ×10{circumflex over ( )}6 CA07 2 monovalent X 1 × 10{circumflex over( )}6 Bris10 3 monovalent X 1 × 10{circumflex over ( )}6 B/WI01 4monovalent X 1 × 10{circumflex over ( )}6 B/Bris60 5 bivalent X X 2 ×10{circumflex over ( )}6 H1H3 6 trivalent X X X 3 × 10{circumflex over( )}6 H3VY 7 trivalent X X X 3 × 10{circumflex over ( )}6 H1VY 8trivalent X X X 3 × 10{circumflex over ( )}6 H1H3V 9 trivalent X X X 3 ×10{circumflex over ( )}6 H1H3Y 10 Quadrivalent X X X X 4 × 10{circumflexover ( )}6 11 Naive none X = component is present in formulation

Six-week-old BALB/c female mice were inoculated intranasally withmonovalent, bivalent, trivalent, or quadrivalent vaccines at doses shownin Table A. A control group of mice was given PBS. Serum samples weretaken on days 7, 14, and 21 after prime inoculation and on days 35, 42and 49 after the second immunization on day 28. Anti-HA IgG antibodytiters from the serum samples were determined by enzyme-linkedimmunosorbent assay (ELISA) against B/Wisconsin/01/2010 andB/Brisbane/60/2008. The humoral response is shown in FIGS. 3A-3B, whichshow that both BM2SR vaccine components (Bris60 and WI01) elevatedanti-influenza virus antibodies higher than the control PBS groupagainst both influenza B antigens representing the two influenza Blineages in multivalent formulations.

These results demonstrate that there is no interference between themonovalent components when formulated into multivalent vaccines.

B. M2SR Mutants Elicit Antibody Responses Against Influenza A VirusFormulated in Multivalent Vaccines

An experiment was performed to demonstrate that M2SR mutant viruseselicit antibody responses when formulated as a monovalent, bivalent,trivalent, or quadrivalent vaccine with the influenza B Yamagata orVictoria lineage BM2SR vaccines. Table A shows how the differentformulations of the following four monovalent M2SR and BM2SR vaccineswere formulated together: A/California/07/2009 (H1N1) (comprising anM2SR-1 mutant comprising SEQ ID NO: 1) A/Brisbane/10/2007 (H3N2)(comprising an M2SR-1 mutant comprising SEQ ID NO: 1),B/Brisbane/60/2008 (Victoria) (comprising a BM2SR-0 mutant comprisingSEQ ID NO: 11), B/Wisconsin/01/2010 (Yamagata) (comprising a BM2SRmutant comprising SEQ ID NO: 11).

Six-week-old BALB/c female mice were inoculated intranasally withmonovalent, bivalent, trivalent, or quadrivalent vaccines at doses shownin Table A. A control group of mice was given PBS. Serum samples weretaken on days 7, 14, and 21 after prime inoculation and on days 35, 42,and 49 after the second immunization on day 28. Anti-HA IgG antibodytiters from the serum samples were determined by enzyme-linkedimmunosorbent assay (ELISA) against A/California/07/2009 (H1N1) andA/Brisbane/10/2007 (H3N2). The humoral response is shown in FIGS. 3C-3D,which shows that both influenza A M2SR vaccine components (H1N1 andH3N2) elevated anti-influenza virus antibodies higher than the controlPBS group against both influenza A antigens representing the H1N1 andH3N2 subtypes in multivalent formulations.

These results demonstrate that there is no interference between themonovalent components when formulated into multivalent vaccines.

Example 3 BM2SR Mutants Protect Mice From Lethal Influenza B VirusChallenge as Monovalent or Quadrivalent Formulations

BALB/c female mice (N=8) were challenged with a lethal dose ofB/Malaysia/2506/2004 virus (20 mouse 50% lethal dose (MLD₅₀)) 49 daysafter the first inoculation (3 weeks after the boost). As shown in FIGS.4A and 4B, all mice vaccinated with the BM2SR and quadrivalent vaccinessurvived the challenge and lost no weight. The control mice that weregiven only PBS, however, lost body weight and did not survive 9 dayspast the challenge date. On day 4 after the challenge, lungs wereobtained and virus titers determined in MDCK cells by plaque assay. Asdepicted in FIG. 4C, lung virus titers in BM2SR and quadrivalentvaccinated mice were below the limit of detection, whereas naïve controlPBS mice had high virus titers indicating that the BM2SR andquadrivalent vaccines confer cross-protection and limit the replicationof the challenge virus.

Example 4 Quadrivalent M2SR Vaccine Protects Mice From Lethal InfluenzaA Virus Challenge

BALB/c female mice (N=8) were challenged with a lethal dose ofA/Aichi/02/1968 (H3N2) virus (40 mouse 50% lethal dose (MLD₅₀)) 49 daysafter the first inoculation (3 weeks after the boost). As shown in FIGS.5A and 5B, all mice vaccinated with the monovalent H1N1 or H3N2 M2SR andquadrivalent M2SR vaccines survived the challenge and lost transientweight but fully recovered. The control mice that were given only PBS,however, lost body weight and did not survive 8 days past the challengedate. On day 4 after the challenge, lungs were obtained and virus titersdetermined in MDCK cells by plaque assay. As depicted in FIG. 5C, lungvirus titers in M2SR monovalents and quadrivalent vaccinated mice wereat least a log lower than naïve control PBS mice indicating that theM2SR monovalents and quadrivalent M2SR vaccines confer cross-protectionand limit the replication of the challenge virus that does not match anyvaccine component.

Example 5 BM2SR Mutants Elicit Antibody Responses Against Influenza BVirus Formulated as Quadrivalent Vaccine

An experiment to demonstrate that BM2SR mutant viruses elicit antibodyresponses when formulated as a quadrivalent vaccine was performed. Thefollowing four monovalent vaccines were formulated together: an H1N1influenza A virus comprising an M2SR-1 mutant comprising SEQ ID NO: 1,an H3N2 influenza A virus comprising an M2SR-1 mutant comprising SEQ IDNO: 1, an influenza B virus of Victoria lineage comprising a BM2SR-4mutant comprising SEQ ID NO: 9, and an influenza B virus of Yamagatalineage comprising a BM2SR-4 mutant comprising SEQ ID NO: 9. 0.2-1×10⁶TCID₅₀ of each monovalent were mixed together such that eachquadrivalent dose was ˜3×10⁶ TCID₅₀ per mouse. The sequence of each ofthe M2SR-1 and BM2SR-4 mutant constructs is provided in Tables 1 and 5.

Six-week-old BALB/c female mice were inoculated intranasally with thequadrivalent formulation at a dose of ˜3×10⁶ TCID₅₀ per mouse. A controlgroup of mice was given PBS. Serum samples were taken on day 14 afterprime inoculation. Anti-HA IgG antibody titers from the serum sampleswere determined by enzyme-linked immunosorbent assay (ELISA) againstinfluenza antigens (B/Victoria and B/Yamagata lineages). As shown inFIGS. 6A and 6B, the quadrivalent M2SR and BM2SR vaccine elevatedanti-influenza virus antibodies higher than the control PBS groupagainst both influenza B antigens representing the two influenza Blineages.

These results demonstrate that each monovalent BM2SR vaccine is capableof eliciting antigen specific responses in a quadrivalent formulation.

Example 6 M2SR and BM2SR Mutants Elicit Antibody Responses AgainstInfluenza A and Influenza B Viruses Formulated in Multivalent Vaccines

A. BM2SR Mutants Elicit Antibody Responses Against Influenza B VirusFormulated in Multivalent Vaccines

An experiment to demonstrate that BM2SR mutant viruses elicit antibodyresponses when formulated as a monovalent, trivalent, or quadrivalentvaccine with the influenza A H1N1 or H3N2 M2SR vaccines is performed.The following four monovalent M2SR and BM2SR vaccines are formulatedtogether: H1N1 (comprising an M2SR-1 mutant comprising SEQ ID NO: 1,H3N2 (comprising an M2SR-1 mutant comprising SEQ ID NO: 1),B/Victoria-lineage (comprising a BM2SR-4 mutant comprising SEQ ID NO:9), B/Yamagata (comprising a BM2SR-4 mutant comprising SEQ ID NO: 9).

Six-week-old BALB/c female mice are inoculated intranasally withmonovalent, trivalent, or quadrivalent vaccines. A control group of micewas given PBS. Serum samples were taken on day 14 after primeinoculation. Anti-HA IgG antibody titers from the serum samples aredetermined by enzyme-linked immunosorbent assay (ELISA) against bothinfluenza B antigens. As shown in FIGS. 7C and 7D, both BM2SR vaccinecomponents were higher than the control PBS group against influenza Bantigens representing the two influenza B lineages in multivalentformulations.

These results demonstrate that there is no interference between themonovalent components when formulated into multivalent vaccines.

B. M2SR Mutants Elicit Antibody Responses Against Influenza A VirusFormulated in Multivalent Vaccines

An experiment to demonstrate that M2SR mutant viruses elicit antibodyresponses when formulated as a monovalent, trivalent, or quadrivalentvaccine with the influenza B Yamagata or Victoria lineage BM2SR vaccineswas performed. The following four monovalent M2SR and BM2SR vaccineswere formulated together: H1N1 (comprising an M2SR-1 mutant comprisingSEQ ID NO: 1), H3N2 (comprising an M2SR-1 mutant comprising SEQ ID NO:1), B/Victoria-lineage (comprising a BM2SR-4 mutant comprising SEQ IDNO: 9), B/Yamagata (comprising a BM2SR-4 mutant comprising SEQ ID NO:9).

Six-week-old BALB/c female mice were inoculated intranasally withmonovalent, trivalent, or quadrivalent vaccines. A control group of micewas given PBS. Serum samples were taken on day 14 after primeinoculation. Anti-HA IgG antibody titers from the serum samples weredetermined by enzyme-linked immunosorbent assay (ELISA) against H1N1 andH3N2 influenza A virus. As shown in FIG. 7A and 7B, both influenza AM2SR vaccine components (H1N1 and H3N2) elevated anti-influenza virusantibodies higher than the control PBS group against both influenza Aantigens representing the H1N1 and H3N2 subtypes in multivalentformulations.

These results demonstrate that there is no interference between themonovalent components when formulated into multivalent vaccines.

Example 7 BM2SR-4 Mutants Protect Mice From Lethal Influenza B VirusChallenge as Monovalent, Trivalent, or Quadrivalent Formulations

BALB/c female mice (N=4) were challenged with a lethal dose of aheterosubtypic influenza B virus, B/Malaysia/2506/2004 virus (20 mouse50% lethal dose (MLD₅₀)), 22 days after the inoculation. All micevaccinated with the BM2SR-4 monovalent, trivalent, and quadrivalentvaccines survived the challenge (FIG. 8B) and lost no weight (FIG. 8A).The control mice that were given only PBS, however, lost body weight anddid not survive challenge. These results indicate that the monovalentBM2SR-4 vaccines (each one different than challenge virus), trivalentand quadrivalent vaccines confer cross-protection against the challengevirus. These results demonstrate that there is no interference betweenthe monovalent components in multivalent formulations.

Example 8 Quadrivalent M2SR Vaccine Protects Mice From Lethal InfluenzaA Virus Challenge

BALB/c female mice (N=4) were challenged with a lethal dose of aheterologous influenza A virus, such as A/Aichi/02/1968 (H3N2) virus (40mouse 50% lethal dose (MLD₅₀)), 22 days after inoculation. All micevaccinated with the trivalent (comprising H1N1 (comprising an M2SR-1mutant comprising SEQ ID NO: 1), H3N2 (comprising an M2SR-1 mutantcomprising SEQ ID NO: 1), and B/Yamagata (comprising a BM2SR-4 mutantcomprising SEQ ID NO: 9)) or quadrivalent M2SR vaccines (comprising H1N1(comprising an M2SR-1 mutant comprising SEQ ID NO: 1), H3N2 (comprisingan M2SR-1 mutant comprising SEQ ID NO: 1), B/Victoria lineage(comprising a BM2SR-4 mutant comprising SEQ ID NO: 9), and B/Yamagata(comprising a BM2SR-4 mutant comprising SEQ ID NO: 9)) had moresurvivors after challenge (FIG. 9B) and lost transient weight butstarted recovery on day 7 (FIG. 9A). The control mice given only PBS,however, lost body weight and did not survive 7 days past the challengedate. These results demonstrate that trivalent and quadrivalentM2SR/BM2SR vaccines confer cross-protection against the challenge virusthat does not match any vaccine component.

Example 9 Immune Responses Elicited by Monovalent M2SR and BM2SR andQuadrivalent M2SR Vaccines and Protective Efficacy in the Ferret ModelA. Summary

This example demonstrates that the immune responses elicited by theQuadrivalent M2SR vaccine are similar to each of the monovalent M2SR andBM2SR vaccines in the ferret model. That is, the Quadrivalent M2SR doesnot display interference and elicits protective immune responses againsteach of the components. Each of the M2SR and BM2SR candidate viruseswere administered intranasally to 12 male ferrets at a dose level of1×10⁷ TCID₅₀ (monovalents) or 4×10⁷ TCID₅₀ (quadrivalent). As a control,one group of ferrets was administered OPTI-MEM™ as a placebo control. Aprime-boost vaccination regimen was utilized for each treatment group.Ferrets were administered the prime vaccine (day 0) and the boostvaccination 28 days later (day 28). Following each vaccination, ferretswere observed for 14 days post inoculation for mortality, with bodyweights, body temperatures and clinical signs measured daily. Serum wascollected on days 21, 35, and 56 from all ferrets post-vaccination toevaluate antibody levels over time.

All animals were challenged intranasally on Day 70 with 1×10⁶ PFU ofA/California/07/2009 (H1N1pdm). Following challenge, ferrets weremonitored for 14 days post inoculation for mortality, with body weights,body temperatures, and clinical signs measured daily. Nasal washes werecollected on days 1, 3, 5, and 7 post challenge from ferrets (N=8) ineach group for viral titers. Additionally, serum was collectedpost-challenge (day 82) from surviving ferrets for analysis. Necropsywas performed on 4 ferrets per group 3 days (day 73) post challenge.Organs were collected for determination of viral load (titers) afterchallenge.

No vaccine-related adverse events were observed among the 5 groups.After challenge, the placebo control group exhibited a reduction (˜15%)in weight. A reduction in weight was also observed in the antigenicallymismatched monovalent H3N2 M2SR and BM2SR vaccinated groups; however,the reduction (˜5-8%) was less than that observed in the placebo group.The Quadrivalent M2SR and H1N1pdm M2SR did not display any significantweight loss after challenge.

B. Materials and Methods

Vaccine Virus Inoculation. Ferrets were inoculated intranasally witheither two doses of a monovalent M2SR or BM2SR vaccine at a dose of1×10⁷ TCID₅₀ or inoculated intranasally with two doses of a quadrivalentM2SR vaccine at a dose of 4×10⁷ TCID₅₀ as shown in Table B. A vial offrozen stock was thawed at room temperature for at least 10 minutes andthen stored refrigerated (or on wet ice) until use. Ferrets wereanesthetized with ketamine/xylazine and the virus dose administeredintranasally in a volume of 500 μL (250 μL per nare). Animals wereobserved daily for 7 days after each vaccination. Body weights, bodytemperatures, and clinical signs were monitored for 7 days.

TABLE B Vaccination and sample collection schedule Nasal Organs³ VaccineDose Vaccination Challenge Washes² n = 3 Serum Group Virus N (TCID₅₀)¹(days) (day) (days) (day) collections⁴ 1 Vehicle 12 N/A 0, 28 70 71, 73,73 21, 35, (Control) 75, 77 56, 82 2 H1N1 12 10⁷ 0, 28 70 71, 73, 73 21,35, M2SR 75, 77 56, 82 3 H3N2 12 10⁷ 0, 28 70 71, 73, 73 21, 35, M2SR75, 77 56, 82 4 B/Bris 12 10⁷ 0, 28 70 71, 73, 73 21, 35, BM2SR 75, 7756, 82 5 BA/Wisc 12 10⁷ 0, 28 70 71, 73, 73 21, 35, BM2SR 75, 77 56, 826 Quad 12 4 × 10⁷ 0, 28 70 71, 73, 73 21, 35, M2SR 75, 77 56, 82¹Inoculated intranasally ²Nasal Washes collected from animals notassigned for necropsy. ³Organs (nasal turbinate, trachea, lung (left andright cranial and caudal lobes) collected from 4 ferrets per group forviral titer analysis. ⁴Post vaccination serum collections

The M2SR virus is a recombinant influenza A virus that does not expressa functional M2 protein, comprising an M2SR-1 mutant comprising SEQ IDNO: 1, encoding the HA and NA genes of Influenza A/Brisbane/10/2007-likeA/Uruguay/716/2007(H3N2) or A/California/07/2009 (H1N1pdm). The BM2SRvirus is a recombinant influenza B virus that does not express afunctional BM2 protein, comprising a BM2SR-0 mutant comprising SEQ IDNO: 11, encoding the HA and NA of B/Brisbane/60/2008 (Victoria) orB/Wisconsin/01/2010 (Yamagata). The Quadrivalent M2SR is composed of 2M2SR and 2 BM2SR viruses that encode for H1N1, H3N2, B/Victoria,B/Yamagata HA and NA.

Animals and Animal Care. Eighty male ferrets were purchased from TripleF Farms and 72 of the ferrets were placed on study. Animals wereapproximately 4 months of age at the time of study initiation. Theanimals were certified by the supplier to be healthy and free ofantibodies to infectious diseases. Upon arrival the animals were singlehoused in suspended wire cages with slat bottoms, suspended overpaper-lined waste pans. The animal room and cages had been cleaned andsanitized prior to animal receipt, in accordance with accepted animalcare practices and relevant standard operating procedures. CertifiedTeklad Global Ferret Diet #2072 (Teklad Diets, Madison Wis.) and city ofChicago tap water were provided ad libitum and were refreshed at leastthree time per week. Fluorescent lighting in the animal rooms wasmaintained on a 12-hr light/dark cycle. Animal room temperature andrelative humidity were within respective protocol limits and ranged from20.0 to 25.0° C. and 30 to 63%, respectively, during the study.

Animal Quarantine and Randomization. The ferrets were held in quarantinefor seven days prior to randomization and observed daily. Based on dailyobservations indicating general good health of the animals, the ferretswere released from quarantine for randomization and testing. Followingquarantine, ferrets were weighed and assigned to treatment groups usinga computerized randomization procedure based on body weights thatproduced similar group mean values [ToxData® version 2.1.E.11 (PDSPathology Data Systems, Inc., Basel, Switzerland)]. Within a group, allbody weights were within 20% of their mean. Animals selected for thestudy receive a permanent identification number by ear tag andtransponder and individual cage cards also identified the study animalsby individual numbers and group. The identifying numbers assigned wereunique within the study.

Experimental Design. To assess the vaccine efficacy, ferrets wereimmunized with each M2SR, BM2SR, or Quadrivalent M2SR virus or mockimmunized by medium (OPTI-MEM™). Ferret body weight, body temperature,and clinical symptoms were monitored and immunological responsesevaluated. 72 male ferrets (Triple F Farms, Sayre Pa.), 4 months of ageat the time of study initiation, were utilized for the study. All animalprocedures were performed in an animal biosafety level-2 facility inaccordance with the protocols approved by the animal care and usecommittee at IIT Research Institute. Prior to inoculation, ferrets weremonitored for 3 days to measure body weight and establish baseline bodytemperatures. Temperature readings were recorded daily through atransponder (BioMedic data systems, Seaford, Del.) implantedsubcutaneously in each ferret. Blood was collected prior to studyinitiation, and serum tested for influenza antibodies. Pre-vaccinationserum samples were treated with receptor destroying enzyme (RDE) toremove nonspecific inhibitors, then serially diluted, tested against adefined amount of influenza virus A/California/07/2009-like (H1N1pdm),A/Switzerland/9715293/2013 (H3N2), Influenza B Virus, B/Brisbane/60/2008(Victoria Lineage) and B/Wisconsin/01/2010 (Yamagata Lineage) and mixedwith 0.5% turkey red blood cells or 0.75-1.0% guinea pig red bloodcells. Antibody titers are defined by the lowest serum dilution causinginhibition of red blood cell agglutination. Only ferrets with HAI(hemagglutination inhibition) titers less than 40 were consideredseronegative and used in this study. Study animals were randomized anddivided into 6 groups (12 ferrets/group) as shown in Table B.

Ferrets were inoculated intranasally with a single dose of 1×10⁷ TCID₅₀of M2SR or BM2SR virus on days 0 and 28, or a single dose of 4×10⁷TCID₅₀ of Quadrivalent M2SR on days 0 and 28. Control group was mockinoculated intranasally with OPTI-MEM™ on days 0 and 28. Ferret bodytemperatures, weights, and clinical symptoms were monitored daily for 14days post-inoculations. Nasal wash samples were kept at −65° C. Bloodwas collected prior to inoculation (day −3 to −5) and days 21, 35 and 56and serum kept at −65° C. until measurement of antibody titer by ELISAand HAI assay.

C. Results

Anti-HA IgG antibody titers from the serum samples were determined byenzyme-linked immunosorbent assay (ELISA) against A/Brisbane/10/2007(H3N2), A/California/07/2009 (H1N1pdm), B/Wisconsin/01/2010 (Yamagatalineage), and B/Brisbane/60/2008 (Victoria lineage). Briefly, ELISAplates were coated by recombinant HA protein from each strain, blockedby bovine serum albumin (BSA), and samples were applied. Ferret IgGantibodies were detected by horseradish peroxidase labeled anti-ferretIgG-goat antibodies (KPL, Inc., Gaithersburg, Md.) and SureBlue TMB(KPL, Inc.) substrate.

As expected, ferrets in each of the immunized groups showed significantelevation of anti-HA antibody in serum to its respective antigen. Moreimportantly, the Quadrivalent M2SR groups demonstrated significantelevation of anti-HA antibody in serum against all four antigens (FIG.10) indicating that there is no interference between the components ofthe multivalent formulation. These data suggest that the M2SR, BM2SR,and Quadrivalent M2SR viruses elicit significant immune responses inferrets.

Serum samples were analyzed by Hemagglutination Inhibition (HAI) assayto demonstrate functional activity of the antibodies detected by ELISA.Serum samples were treated with receptor-destroying enzyme (RDE) (DenkaSeiken, Tokyo, Japan) to eliminate inhibitors of nonspecifichemagglutination. RDE was reconstituted per the manufacturer'sinstructions. Serum was diluted 1:3 in RDE and incubated 18-20 hours ina 37° C.±2° C. water bath. After the addition of an equal volume of 2.5%(v/v) sodium citrate, the samples were incubated in a 56±2° C. waterbath for 30±5 minutes. 0.85% NaCl was added to each sample to a finalserum dilution of 1:10 after the RDE treatment. The diluted samples werethen diluted into four two-fold dilutions (1:10 to 1:80) in duplicate inphosphate buffered saline (PBS) then incubated with 4 hemagglutinatingunits of A/Brisbane/10/2007 (H3N2), A/California/07/2009 (H1N1pdm),B/Wisconsin/01/2010 (Yamagata lineage) and B/Brisbane/60/2008 (Victorialineage) influenza viruses. After incubation, 0.5% avian red blood cellswere added to each sample and incubated for 30±5 minutes. Presence orabsence of hemagglutination was then scored.

As shown in FIGS. 11A and 11B, all M2SR immunized ferrets demonstratedsignificant HAI antibody titers against their respective test virus. TheQuadrivalent M2SR demonstrated significant HAI titers against all fourtest viruses. The placebo (naïve) group did not elicit any influenzaspecific antibodies. The CDC states that serum HAI antibody titers of 40are associated with at least a 50% reduction in risk for influenzainfection or disease in populations. Therefore, these results suggestthat M2SR and BM2SR viruses elicit protective immune responses that aremaintained when the viruses are formulated together as a Quadrivalentvaccine.

After challenge with A/California/09/2009 (H1N1pdm), a 5-8% loss of bodyweight was observed by Day 6 post challenge in all animals. Throughoutthe 14-day observation period, animal body weights remained below theirinitial weight, except the OPTI-MEM™-administered ferrets (placebogroup) lost the most weight (15%). Weight loss among vaccinated ferretswas dependent on the antigenicity of the vaccine. Ferrets receiving thematching H1N1pdm M2SR or the Quadrivalent M2SR (that contains theH1N1pdm M2SR) did not display any significant weight loss. Ferretsreceiving a heterologous H3N2 M2SR or either of the BM2SR vaccinesdisplayed ˜5-8% weight loss.

Nasal wash samples were collected from all ferrets on days 1, 3, 5, 7post-challenge and evaluated for the presence of challenge virus byplaque assay in MDCK cells. FIG. 12 shows that the Quadrivalent M2SRcontrolled challenge virus replication in a manner similar to themonovalent homologous H1N1pdm M2SR. The placebo and BM2SR monovalentvaccines did not control the challenge virus with at least 5 logs ofvirus being detected up to 5 days post-infection. The heterologous H3N2M2SR group did not eliminate the challenge virus like the homologousH1N1 and Quad M2SR but did partially control virus replication relativeto the placebo group.

Respiratory organs harvested on day 3 post-infection from 4 ferretsdemonstrated the control of challenge virus. The H1N1 M2SR and Quad M2SRdid not allow the challenge virus to replicate in the upper and lowerrespiratory tissues at all (nasal turbinate, trachea, lung) as shown inFIGS. 13A, 13B, and 13C). In contrast, the challenge virus grew to hightiters in the upper respiratory tissues (nasal turbinate and trachea) ofthe other groups. In the lower respiratory tract (lung), the monovalentM2SR vaccines controlled the challenge virus relative to the placebogroup. These results suggest that the homologous and Quadrivalent M2SRprevent influenza infection from establishing itself and that theunrelated M2SR vaccines reduce severity of infection.

D. Conclusion

This example shows that intranasal administration of the QuadrivalentM2SR vaccine virus was not associated with any vaccine-related adverseevents (e.g., elevated body temperature, loss of weight, or clinicalsigns). These results show that the Quadrivalent M2SR virus elicitsprotective immune responses against each strain contained in themultivalent formulation and is useful as an intranasal influenzavaccine.

What is claimed is:
 1. An immunogenic composition, wherein thecomposition is a multivalent composition comprising recombinant virusesfrom at least two influenza strains, wherein the multivalent compositioncomprises: a) at least one engineered attenuated influenza AM2-deficient recombinant virus, wherein the engineered influenza A viruscomprises a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3; and b) at least one engineered attenuated influenzaBM2-deficient recombinant virus, wherein the engineered influenza Bvirus comprises a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:
 11. 2. Theimmunogenic composition of claim 1, wherein the at least one influenza Avirus is chosen from the group of H1N1 and H3N2 subtypes, and the atleast one influenza B virus is chosen from the group of B/Yamagata andB/Victoria lineages.
 3. The immunogenic composition of claim 1, whereinthe multivalent composition comprises recombinant viruses selected fromthe group consisting of: a) two engineered attenuated influenza AM2-deficient viruses chosen from the group of H1N1 and H3N2 subtypes,wherein the A M2-deficient viruses comprise a mutant M2 gene comprisingSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and two engineeredattenuated influenza BM2-deficient viruses chosen from the group ofB/Yamagata and B/Victoria lineages, wherein the BM2-deficient virusescomprise a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:11; b) two engineeredattenuated influenza A M2-deficient viruses chosen from the group ofH1N1 and H3N2 subtypes, wherein the A M2-deficient viruses comprise amutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3,and one engineered attenuated influenza BM2-deficient virus chosen fromthe group of B/Yamagata and B/Victoria lineages, wherein theBM2-deficient virus comprises a mutant BM2 gene comprising SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ IDNO:11; c) one engineered attenuated influenza A M2-deficient viruschosen from the group of H1N1 and H3N2 subtypes, wherein the AM2-deficient virus comprises a mutant M2 gene comprising SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3, and two engineered attenuated influenzaBM2-deficient viruses chosen from the group of B/Yamagata and B/Victorialineages, wherein the BM2-deficient viruses comprise a mutant BM2 genecomprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, or SEQ ID NO:11; and d) one engineered attenuated influenza AM2-deficient virus chosen from the group of H1N1 and H3N2 subtypes,wherein the A M2-deficient virus comprises a mutant M2 gene comprisingSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and one engineeredattenuated influenza BM2-deficient virus chosen from the group ofB/Yamagata and B/Victoria lineages, wherein the BM2-deficient viruscomprises a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:11.
 4. Theimmunogenic composition of claim 1, wherein the multivalent compositionis a quadrivalent composition comprising: a) two engineered attenuatedinfluenza A viruses consisting of: i) H1N1 having a mutant M2 genecomprising SEQ ID NO: 1, and ii) H3N2 having a mutant M2 gene comprisingSEQ ID NO: 1; and b) two engineered attenuated influenza B virusesconsisting of: i) B/Victoria having a mutant BM2 gene comprising SEQ IDNO: 9 or SEQ ID NO: 11, and ii) B/Yamagata having a mutant BM2 genecomprising SEQ ID NO: 9 or SEQ ID NO:
 11. 5. The immunogenic compositionof claim 1, wherein the multivalent composition is a quadrivalentcomposition comprising: a) two engineered attenuated influenza A virusesconsisting of: i) H1N1 having a mutant M2 gene comprising SEQ ID NO: 1,and ii) H3N2 having a mutant M2 gene comprising SEQ ID NO: 1; and b) oneengineered attenuated influenza B viruses selected from the groupconsisting of: i) B/Victoria having a mutant BM2 gene comprising SEQ IDNO: 9 or SEQ ID NO: 11, and ii) B/Yamagata having a mutant BM2 genecomprising SEQ ID NO: 9 or SEQ ID NO:
 11. 6. The immunogenic compositionof claim 1, further comprising a pharmaceutically acceptable carrier. 7.The immunogenic composition of claim 1, further comprising apharmaceutically acceptable adjuvant.
 8. The immunogenic composition ofclaim 1, wherein the composition is formulated for intranasal orintracutaneous administration.
 9. A method of stimulating an immuneresponse against influenza A and influenza B comprising administering toa subject in need thereof a multivalent immunogenic compositioncomprising: a) at least one engineered attenuated influenza AM2-deficient recombinant virus, wherein the engineered influenza A viruscomprises a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3; and b) at least one engineered attenuated influenzaBM2-deficient recombinant virus, wherein the engineered influenza Bvirus comprises a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:
 11. 10. Themethod of claim 9, wherein the at least one influenza A virus is chosenfrom the group of H1N1 and H3N2 subtypes, and the at least one influenzaB virus is chosen from the group of B/Yamagata and B/Victoria lineages.11. The method of claim 9, wherein the multivalent immunogeniccomposition comprises recombinant viruses selected from the groupconsisting of: a) two engineered attenuated influenza A M2-deficientviruses chosen from the group of H1N1 and H3N2 subtypes, wherein the AM2-deficient viruses comprise a mutant M2 gene comprising SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3, and two engineered attenuated influenzaBM2-deficient viruses chosen from the group of B/Yamagata and B/Victorialineages, wherein the BM2-deficient viruses comprise a mutant BM2 genecomprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, or SEQ ID NO:11; b) two engineered attenuated influenza AM2-deficient viruses chosen from the group of H1N1 and H3N2 subtypes,wherein the A M2-deficient viruses comprise a mutant M2 gene comprisingSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and one engineeredattenuated influenza BM2-deficient virus chosen from the group ofB/Yamagata and B/Victoria lineages, wherein the BM2-deficient viruscomprises a mutant BM2 gene comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:11; c) oneengineered attenuated influenza A M2-deficient virus chosen from thegroup of H1N1 and H3N2 subtypes, wherein the A M2-deficient viruscomprises a mutant M2 gene comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3, and two engineered attenuated influenza BM2-deficient viruseschosen from the group of B/Yamagata and B/Victoria lineages, wherein theBM2-deficient viruses comprise a mutant BM2 gene comprising SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ IDNO:11; and d) one engineered attenuated influenza A M2-deficient viruschosen from the group of H1N1 and H3N2 subtypes, wherein the AM2-deficient virus comprises a mutant M2 gene comprising SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 3, and one engineered attenuated influenzaBM2-deficient virus chosen from the group of B/Yamagata and B/Victorialineages, wherein the BM2-deficient virus comprises a mutant BM2 genecomprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, or SEQ ID NO:11.
 12. The method of claim 9, wherein themultivalent immunogenic composition is a quadrivalent compositioncomprising: a) two engineered attenuated influenza A viruses consistingof: i) H1N1 having a mutant M2 gene comprising SEQ ID NO: 1, and ii)H3N2 having a mutant M2 gene comprising SEQ ID NO: 1; and b) twoengineered attenuated influenza B viruses consisting of: i) B/Victoriahaving a mutant BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11, andii) B/Yamagata having a mutant BM2 gene comprising SEQ ID NO: 9 or SEQID NO:
 11. 13. The method of claim 9, wherein the multivalentimmunogenic composition is a quadrivalent composition comprising: a) twoengineered attenuated influenza A viruses consisting of: i) H1N1 havinga mutant M2 gene comprising SEQ ID NO: 1, and ii) H3N2 having a mutantM2 gene comprising SEQ ID NO: 1; and b) one engineered attenuatedinfluenza B viruses selected from the group consisting of: i) B/Victoriahaving a mutant BM2 gene comprising SEQ ID NO: 9 or SEQ ID NO: 11, andii) B/Yamagata having a mutant BM2 gene comprising SEQ ID NO: 9 or SEQID NO:
 11. 14. The method of claim 9, wherein the immunogeniccomposition further comprises a pharmaceutically acceptable carrier. 15.The method of claim 9, wherein the immunogenic composition furthercomprises a pharmaceutically acceptable adjuvant.
 16. The method ofclaim 9, wherein the immunogenic composition is formulated forintranasal or intracutaneous administration.